Long lasting drug formulations

ABSTRACT

The present invention is directed to long-lasting erythropoietin therapeutic formulations and their methods of use wherein the formulation comprises a genetically modified micro-organ that comprises a vector which comprises a nucleic acid sequence operably linked to one or more regulatory sequences, wherein the nucleic acid sequence encodes erythropoietin.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 13/346,761, filedon Jan. 10, 2012 (and entitled LONG LASTING DRUG FORMULATIONS), which isa Continuation in Part of U.S. application Ser. No. 13/160,632 filedJun. 15, 2011, which claims priority from U.S. Provisional ApplicationSer. No. 61/355,029 filed Jun. 15, 2010 and U.S. Provisional ApplicationSer. No. 61/414,921 filed Nov. 18, 2010; which is a Continuation in Partapplication of U.S. application Ser. No. 11/898,481, filed Sep. 12,2007, which claims priority from U.S. Provisional Application Ser. No.60/844,351, filed Sep. 14, 2006; all of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

This invention is directed to long-lasting therapeutic formulationscomprising a genetically modified micro-organ comprising a vectorcomprising a nucleic acid sequence encoding a therapeutic polypeptide,such as erythropoietin, operably linked to one or more regulatorysequences and their methods of use.

BACKGROUND OF THE INVENTION

Therapeutic agents can be delivered orally, transdermally, byinhalation, by injection or by depot with slow release. However, themethod of delivery is limited by the processing that the agent issubjected to in the recipient, by the requirement for frequentadministration, and limitations on the size of molecules that can beutilized. For some of the methods, the amount of therapeutic agentvaries between administrations.

Protein production techniques which involve the sub-cloning of a desirednucleic acid sequence/fragment into a vector which is subsequently usedfor modifying specific host cells, which are meant to produce thedesired protein for further purification steps are limited in the amountof protein expressed, protein secretion, post-translationalmodifications (such as glycosylation and the accurate folding of theprotein), etc. Moreover, even if a high-level of protein productioncould be achieved, large quantities of the recombinant protein must thenbe produced and purified to be free of contaminants. Development of apurification scheme is a very lengthy process. And once purifiedrecombinant protein has been obtained, it must be further formulated torender it stable and acceptable for introduction into animals or humans.Furthermore, even formulated, purified recombinant proteins have afinite shelf life due to maintenance and storage limitations; oftenrequiring repeated purification and formulation of more protein. Theprocess of developing an appropriate formulation is time consuming,difficult, and costly, as well.

Thus, there is a widely recognized need for long-lasting protein-basedtherapeutic molecules that have the requisite post-translationalmodifications to preserve their biological activity, which are producedinexpensively and quickly without the need for the laborious and costlymethods typically associated with obtaining high-levels of recombinantproteins.

Some researchers have attempted to obtain in vivo expression ofrecombinant gene products via gene therapy. Typically viral vectors areused to transduce cells in vivo to express recombinant gene products.These viral-based vectors have advantageous characteristics, such as thenatural ability to infect the target tissue. However, retrovirus-basedvectors require integration within the genome of the target tissue toallow for recombinant product expression (with the potential to activateresident oncogenes) and can only be used to transduce actively dividingtissues. Viral vectors are also often not able to sustain long-termtransgene expression, which may be due at least in part to theirelimination due to secondary host immune responses.

Accordingly, there remains a need in the art for recombinant geneproduct formulations that have consistently high expression levelslasting for several weeks or more and for methods of using thoseformulations to treat disease.

SUMMARY OF THE INVENTION

The invention provides, in one embodiment, a long-lasting therapeuticformulation comprising a genetically modified micro-organ, saidmicro-organ comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences, wherein saidnucleic acid sequence encodes a therapeutic polypeptide and wherebyadministration of said formulation increases blood hemoglobin (“Hb”)levels over basal level and said increase is maintained for at least onemonth. In one embodiment, the vector is a helper-dependent adenovirusvector.

In one embodiment, the therapeutic polypeptide is erythropoietin. In oneembodiment, the erythropoietin is encoded by SEQ ID No. 7. In oneembodiment, this invention provides a long-lasting erythropoietinformulation comprising at least one genetically modified micro-organthat expresses and secretes erythropoietin, said micro-organ comprisinga helper-dependent adenovirus vector, said vector comprising a nucleicacid sequence encoding erythropoietin operably linked to one or moreregulatory sequences, wherein said nucleic acid comprises SEQ ID No. 11.

In another embodiment, the invention provides a method of providing atherapeutic polypeptide to a subject in need over a sustained periodcomprising providing one or more genetically modified micro-organs, saidmicro-organs comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences; and implanting saidgenetically modified micro-organ in said subject, wherein said nucleicacid sequence encodes a therapeutic polypeptide and whereby saidformulation increases Hb levels over the basal level and said increaseis maintained for at least one month. In yet another embodiment, theincrease is maintained for greater than one month. In one embodiment,the Hb levels are maintained within a range of 9-11 g/dl hemoglobin. Inanother embodiment, the Hb levels are maintained within a range of 10-12g/dl. In one embodiment, the Hb levels are maintained for at least onemonth.

In one embodiment, the vector is a helper-dependent adenovirus vector.In one embodiment, the therapeutic polypeptide is erythropoietin. In oneembodiment, the erythropoietin is encoded by SEQ ID No. 7. In oneembodiment, the subject in need is suffering from anemia. In anotherembodiment, the subject in need is suffering from an infection. In yetanother embodiment, the subject in need is suffering from cancer.

In one embodiment, this invention provides a method of treating anemiain a human subject in need over a sustained time period comprising thesteps of: a. providing at least one genetically modified micro-organthat expresses and secretes erythropoietin, wherein the micro-organcomprises a helper-dependent adenovirus vector comprising a nucleic acidsequence encoding erythropoietin operably linked to one or moreregulatory sequences; b. determining erythropoietin secretion levels ofthe at least one genetically modified micro-organ in vitro; c.implanting the at least one genetically modified micro-organ in thehuman subject at an effective dosage; and d. measuring hemoglobin levelsin the blood of the subject, wherein the measured hemoglobin levels inthe subject are maintained at 9-11 g/dl in at least 50% of themeasurements for at least one month.

In one embodiment, this invention provides an effective dosage of 18-150IU erythropoietin/Kg bodyweight of said subject/day. In one embodiment,the effective dosage is determined based on a subject's weight,historical hemoglobin levels and the average amount of erythropoietinpreviously administered to the subject for one month prior to saidimplanting step.

In one embodiment, this invention provides a method of increasing ormaintaining physiological hemoglobin levels in a human subject over asustained period of time comprising the steps of: a. providing at leastone genetically modified micro-organ that expresses and secreteserythropoietin, the micro-organ comprising a helper-dependent adenovirusvector comprising a nucleic acid sequence encoding erythropoietinoperably linked to one or more regulatory sequences; b. determiningerythropoietin secretion levels of the at least one genetically modifiedmicro-organ in vitro; c. implanting the at least one geneticallymodified micro-organ in the subject at an effective dosage; and d.measuring hemoglobin levels in the blood of the subject, wherein theincreased or maintained physiological hemoglobin levels in said subjectare maintained at 9-11 g/dl in at least 50% of the measurements for atleast one month.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents levels of recombinant optimized human interferon-alpha(IFNα) produced in vitro by the formulations of the instant invention.

FIG. 2 presents levels of recombinant human erythropoietin (hEPO)produced in vitro by the formulations of the instant invention.HD-Ad-CAG-wt-hEPO GMMO titration (A). Micro-organs were transduced withincreasing dilutions of HD-Ad-CAG-wt-hEPO virus: 1:25; 1:100; and 1:1000dilutions. Ad5/CMV/wt-hEPO was diluted to a working concentration of1:10 and 1:50. A comparison between GMMOs produced from two differentskins, H-1 and H-2 (B). Micro-organs were transduced withHD-Ad-CAG-wt-hEPO 1:25. Bars indicate the hEPO concentration measured byELISA in the culture media that was collected and replaced every 3-4days.

FIG. 3 presents the percent of peak erythropoietin (EPO) expressionlevels in vitro from optimized formulations comprising EPO-expressinggutless adenovirus and micro-organs comprising EPO-expressingadenovirus-5. Micro-organs were transduced with HD-Ad-CAG-hEPO at 1:25or with Ad5/CMV/hEPO at 1:10.

FIG. 4 presents erythropoietin (EPO) expression levels in vitro fromformulations comprising optimized and non-optimized EPO-expressinggutless adenovirus. Micro-organs were transduced with a working dilutionof 1:100 viral particles. Bars indicate the hEPO concentration measuredby ELISA in the culture media that was collected and replaced every 3-4days.

FIG. 5 presents erythropoietin (EPO) expression levels in vitro fromformulations comprising EPO-expressing gutless adenovirus downstream ofa CAG or CMV promoter.

FIG. 6 presents levels of recombinant human erythropoietin produced invivo in SCID mice (A) and in vitro (B) by the formulations of theinstant invention in vitro and the associated changes in hematocrit (A).Ten mice/group were implanted subcutaneously with GMMOs. The hEPO levels(mU/ml) and the corresponding % hematocrit that were measured in theserum of mice that were implanted with GMMOs transduced withadenovirus-hEPO, helper-dependent adenovirus-hEPO, and helper-dependentadenovirus-optimized hEPO and with non-transduced GMMOs are presented.Bleeds were done every 10 days (A). Hematocrit was measured by thecentrifugation method and serum hEPO levels in the blood were measuredby a hEPO ELISA kit. Non-implanted GMMOs were maintained in culture andlevels of EPO were measured (B).

FIG. 7 presents the correlation of peak serum EPO levels to delivereddose in vivo, in human patients.

FIG. 8A presents summary results of hEPO-GMMO low dose clinical group,illustrating a sustained hemoglobin response within a therapeutic window(10-12 g/dl) in 5 of 6 patients for periods of 1-36 months withoutinjections. FIG. 8B presents summary results of hEPO-GMMO mid doseclinical group, illustrating a sustained hemoglobin response within thetherapeutic window (10-12 g/dl) in 6 of 7 patients, for periods of 3-18months without injections. FIG. 8C presents summary results of hEPO-GMMOhigh dose clinical group, illustrating a sustained hemoglobin responsewithin the therapeutic window (10-12 g/dl) in 3 patients for periods of1-4 months without injections.

FIG. 9 presents hemoglobin response of Patient 1.

FIG. 10A presents sustained hemoglobin response of Patient 2, FIG. 10Bpresents reticulocytes count in response to hEPO-GMMO administration inPatient 2, and FIG. 10C presents serum EPO levels in response tohEPO-GMMO in Patient 2.

FIG. 11A presents hemoglobin response of Patient 3. FIG. 11B presentshemoglobin levels prior to hEPO-GMMO implantation with the projectednadir.

FIG. 12 presents sustained hemoglobin response of Patient 4.

FIG. 13 presents sustained hemoglobin response of Patient 6.

FIG. 14 presents sustained hemoglobin response of Patient 7.

FIG. 15 presents base-line demographics of clinical study patients.

FIG. 16 presents hemoglobin and serum EPO levels following doseaugmentation by a second administration.

FIG. 17 presents an expression cassette for HDAd-hEPO vector.

FIG. 18 illustrates schematically construction of pΔ28E4-CAG-EPOplasmid.

FIG. 19 illustrates schematically construction/production of theHDAd-EPO vector.

FIG. 20 presents EPO secretion ex vivo from transduced micro-organsobtained from different subjects.

FIG. 21 presents GMMO-EPO viability by cell count at different timesduring maintenance.

FIG. 22 presents dermal GMMO-EPO titration analysis.

FIG. 23 presents sustained long-lasting in vitro EPO secretion.

FIG. 24 presents elevated serum EPO and subsequent rise in hematocrit inSCID mice.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In some embodiments, the instant invention is directed to long-lastingtherapeutic formulations comprising a genetically modified, tissue-basedmicro-organ comprising a vector comprising a nucleic acid sequenceencoding a therapeutic polypeptide, such as erythropoietin, operablylinked to one or more regulatory sequences and their methods of use.

The invention provides, in one embodiment, a long-lasting therapeuticformulation comprising a genetically modified micro-organ, saidmicro-organ comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences, wherein saidnucleic acid sequence encodes a therapeutic polypeptide and wherebyadministration of the therapeutic formulation increases the level ofhemoglobin (“Hb”) and the increase is maintained for at least one month.In another embodiment, the increase is for greater than one month. Inanother embodiment, the hemoglobin level is increased and the increaseis maintained for greater than six months.

In another embodiment, this invention provides a long-lastingtherapeutic formulation comprising a genetically modified micro-organ,said micro-organ comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences, wherein saidnucleic acid sequence encodes a therapeutic polypeptide and wherebyadministration of the formula increases blood Hb levels over basallevels and the increase is maintained for at least one month and whereinthe vector is a helper-dependent adenovirus vector.

In yet another embodiment, this invention provides a long-lastingerythropoietin formulation comprising at least one genetically modifiedmicro-organ that expresses and secretes erythropoietin, the micro-organcomprising a helper-dependent adenovirus vector, the vector comprising anucleic acid sequence encoding erythropoietin operably linked to one ormore regulatory sequences, wherein the nucleic acid encodingerythropoietin operably linked to one or more regulatory sequencescomprises SEQ ID No. 11. Administration of such a formulation at aneffective dosage to a human subject in need can increase and/or maintainhemoglobin levels in the subject to physiological hemoglobin levels forat least one month.

As used herein, the term “subject” refers to a human subject. The term“subject” may also be referred to herein as a “patient”. Subjects may benaïve, e.g., a patient naïve to EPO. Alternatively, subjects may bepreviously exposed to a therapeutic polypeptide, e.g., EPO, for instanceby way of erythroid stimulating activity (ESA; also termederythropoietic stimulating agent) injection therapy.

As used herein, the term “increased hemoglobin levels” refers to anincrease in blood Hb levels over basal levels in response toadministration of a long-lasting therapeutic formulation of the currentinvention to a subject in need. As used herein, the term “increasedhemoglobin levels” may also be referred to herein as “hemoglobinresponse”. Administration of a GMMO to a naïve subject may increasehemoglobin levels to a therapeutic level. Administration of a GMMO to asubject previously exposed to EPO may maintain hemoglobin levels at atherapeutic level.

In one embodiment, the Hb response refers to an increase in Hb levelssuch that Hb levels range between 9-11 gm/dl, which is the current FDArecommended range. In another embodiment, the Hb levels range between9.5-12.6 gm/dl. In yet another embodiment, the Hb levels range between10-12 gm/dl. In still another embodiment, the Hb levels range between9-13.2 gm/dl. In a further embodiment, the Hb levels range between8.5-13.8 gm/dl. In another embodiment, the Hb levels range between8-14.4 gm/dl.

As Hb levels in blood may oscillate slightly from day to day, the rangeincrease in Hb response may in certain situations represent an averageincrease over any given time period. Measurements made over a given timeperiod may reflect this oscillation. For example, the increased Hb maybe maintained for 90% of measurement over any given time period within atarget range, as for example presented above. In other words, 90% ofmeasurements made during at least one month or 90% of measurements madeover at least six months, or at least one year may be within the Hbtarget range. Alternatively, Hb levels may be increased or maintainedwithin the targeted range for 80% of measurements over any given timeperiod. Further, Hb levels may be increased or maintained within thetargeted range for 70% of measurements over any given time period.Alternatively, Hb levels may be increased or maintained within thetargeted range for 60% of measurements over any given time period. Or,Hb levels may be increased or maintained within the targeted range for50% of measurements over any given time period.

Hemoglobin measurements may be made on a regular basis or irregularbasis. In certain cases, measurements of blood Hb levels may be madeonce per week. Alternatively, measurements of blood Hb may be more orless frequent, e.g., twice per week or once every two weeks or once amonth. In one embodiment, blood measurements are made once a week. Inanother embodiment, twice a week. In yet another embodiment, three timesa week. In still another embodiment, measurements are made once everytwo weeks. In a further embodiment, measurements are made once a month.In one embodiment, measurements are made on a regularly scheduled basis.In another embodiment, measurements are made on an as “needed” basis.Measurements may be made more or less frequently, dependent on need.

In some embodiments, increased Hb levels are maintained within a givenrange for at least 90% of the time that Hb levels are increased. Inother embodiments, Hb levels are maintained for at least 80% of thetime. In yet other embodiments, Hb levels are maintained for at least70% of the time. In still other embodiments, Hb levels are maintainedfor at least 60% of the time. In a further embodiment, Hb levels aremaintained for at least 50% of the time.

As used herein, the term “hematocrit” refers to the packed cell volumeor erythrocyte volume fraction as a percentage of the concentration ofred blood cells in blood. As used herein, increases in Hb levels reflectincreases in hematocrit.

For patients previously treated with ESA injection therapy,administration of “human erythropoietin-genetically modifiedmicro-organ” (“hEPO-GMMO”) in place of ESA injections prevents adecrease in Hb levels to their natural nadir. In one embodiment, whereinthe patient has been treated with ESA, Hb response refers to aprevention of the decrease of Hb level that would otherwise occurnaturally and maintenance of elevated Hb levels, compared with thepatient's natural nadir. In one embodiment, hEPO-GMMO administrationprevents a decrease in Hb levels. In this way Hb levels may bemaintained within the therapeutic window.

As used herein, the term “erythropoiesis” refers to the process of redblood cell formation or production. Erythropoietin is a required elementin the regulation of erythropoiesis, i.e., red blood cell production.The measure of an Hb response is also a measure of red blood cellformation, i.e., erythropoiesis.

As used herein, a sustained “hemoglobin response” may also be referredto as sustained “erythropoiesis” having all the qualities and propertiesof an Hb response.

In another embodiment, the invention provides a long-lasting therapeuticformulation comprising a genetically modified micro-organ, saidmicro-organ comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences, wherein saidnucleic acid sequence encodes a therapeutic polypeptide and wherebyblood Hb level is increased over basal level following administration ofthe formulation and the increase is maintained for greater than onemonth in a subject, wherein the subject may be an immuno-competent host.

In one embodiment, the expression level of the nucleic acid is increasedby more than 5% over basal levels in an immuno-competent host, while inanother embodiment, the vector is a helper-dependent adenovirus vector.

The invention provides a long-lasting therapeutic formulation andmethods of use thereof, where the formulation comprises a geneticallymodified micro-organ. The term “micro-organ” as used herein, refers inone embodiment, to an isolated tissue or organ structure derived from oridentical to an explant that has been prepared in a manner conducive tocell viability and function. In one embodiment, a micro-organ maintainsat least some in vivo structures, or in another embodiment,interactions, similar to the tissues or organ from which it is obtained.In one embodiment, a micro-organ is an intact, isolated tissue slice. Inanother embodiment, micro-organs retain the micro-architecture and thethree dimensional structure of the tissue or organ from which they werederived and have dimensions selected so as to allow passive diffusion ofadequate nutrients and gases to cells within the micro-organ anddiffusion of cellular waste out of the cells of the micro-organ so as tominimize cellular toxicity and concomitant cell death due toinsufficient nutrition and/or accumulation of waste. In one embodiment,a micro-organ is a sliver of dermal tissue.

In one embodiment, a micro-organ is 1-2 mm in diameter and 30-40 mm inlength. In another embodiment, the diameter of a micro-organ may be, forexample, 1-3 mm, 1-4 mm, 2-4 mm, 0.5-3.5 mm, 1.5-2.5 or 1.5-10 mm. Inanother embodiment the diameter of a micro-organ may be, for example,approximately 2 mm or approximately 1.5 mm. In another embodiment, thelength of the micro-organ may be 5-100 mm, 10-60 mm, 20-60 mm, 20-50 mm,20-40 mm, 20-100 mm, 30-100 mm, 40-100 mm, 50-100 mm, 60-100 mm, 70-100mm, 80-100 mm, or 90-100 mm. In another embodiment, the length of themicro-organ may be approximately 20 mm, approximately 30 mm,approximately 40 mm, or approximately 50 mm. In one embodiment, amicro-organ is smaller than 1.5 cm², and in another embodiment, lessthan 1 cm². In another embodiment, the diameter is less than 1.5 cm, andin another embodiment, the length is less than 1.5 cm.

In one embodiment, a micro-organ is an explant. In one embodiment, amicro-organ is tissue-derived. In another embodiment, a micro-organ is asection or portion or part of a tissue. In another embodiment, amicro-organ is a section or portion or part of an organ. A micro-organcan be distinguished from a skin graft, in one embodiment, in that it isspecifically designed to survive for long periods of time in vivo and invitro and, in another embodiment, in that its dimensions arespecifically selected so as to allow passive diffusion of adequatenutrients and gases to cells within the micro-organ and diffusion ofcellular waste out of the cells of the micro-organ, which in oneembodiment minimizes cellular toxicity and concomitant cell death due toinsufficient nutrition and/or accumulation of waste. Thus, in oneembodiment, a micro-organ is not a skin graft. In another embodiment, amicro-organ can be distinguished from a collection of isolated cells,which in one embodiment, are grown on a natural or artificial scaffold,in that micro-organs maintain the micro-architecture and the threedimensional structure of the tissue or organ from which they werederived. Thus, in one embodiment, a micro-organ is not one or more celltypes grown on a scaffold or within a gel.

A detailed description of micro-organs can be found in US-2003-0152562,which is incorporated herein by reference in its entirety.

Earlier patent applications (WO 03/006669, WO 03/03585, WO 04/099363,which are incorporated in-full herein by reference) describedmicro-organs, which can be modified to express a gene product ofinterest, that may be sustained outside the body in an autonomouslyfunctional state for an extended period of time, and may then beimplanted subcutaneously or in other locations within the body for thepurpose of treating diseases or disorders. In one embodiment, amicro-organ that is modified to express a gene product of interest is atherapeutic micro-organ. The therapeutic micro-organs of the presentinvention unexpectedly showed a much longer-term expression profile of agene product of interest in vitro and in vivo.

As used herein, the term “explant” refers, in one embodiment, to atissue or organ or a portion thereof removed from its natural growthsite in an organism and placed in a culture medium for a period of time.In one embodiment, the tissue or organ is viable, in another embodiment,metabolically active, or a combination thereof. As used herein, the term“explant” may, in some embodiments, be used interchangeably with“micro-organ” or “micro-organ explant”.

As used herein, the term “microarchitecture” refers, in one embodiment,to a characteristic of the explant in which some or all of the cells ofthe tissue explant maintain, in vitro, physical and/or functionalcontact with at least one cell or non-cellular substance with which theywere in physical and/or functional contact in vivo.

In another embodiment, micro-organ explants maintain thethree-dimensional structure of the tissue or organ from which they werederived. In one embodiment, micro-organ explants retain the spatialinteractions, e.g. cell-cell, cell-matrix and cell-stromal interactions,and the orientation of the tissue from which they were derived. In oneembodiment, preservation of spatial interactions such as described abovepermit the maintenance of biological functions of the explant, such assecretion of autocrine and paracrine factors and other extracellularstimuli, which in one embodiment, provide long term viability to theexplant. In one embodiment, at least some of the cells of themicro-organ explant maintain, in vitro or in vivo after implantation,their physical and/or functional contact with at least one cell ornon-cellular substance with which they were in physical and/orfunctional contact in vivo. In one embodiment, some of the cells refersto at least about 50%, in another embodiment, at least about 60%, inanother embodiment at least about 70%, in another embodiment, at leastabout 80%, and in another embodiment, at least about 90% or more of thecells of the population. In another embodiment, the cells of the explantmaintain at least one biological activity of the organ or tissue fromwhich they are isolated.

In one embodiment, the term “about”, refers to a deviance of between0.0001-5% from the indicated number or range of numbers. In oneembodiment, the term “about”, refers to a deviance of between 1-10% fromthe indicated number or range of numbers. In one embodiment, the term“about”, refers to a deviance of up to 25% from the indicated number orrange of numbers.

In some embodiments, any of the formulation of this invention willcomprise a genetically modified micro-organ (GMMO), in any form orembodiment as described herein. As used herein, the term “GMMO” may alsorefer to a “Biopump”. In some embodiments, any of the formulations ofthis invention will consist of a genetically modified micro-organ, inany form or embodiment as described herein. In some embodiments, of thecompositions of this invention will consist essentially of a geneticallymodified micro-organ, in any form or embodiment as described herein. Insome embodiments, the term “comprise” refers to the inclusion of theindicated active agent, such as the genetically modified micro-organ, aswell as inclusion of other active agents, and pharmaceuticallyacceptable carriers, excipients, emollients, stabilizers, etc., as areknown in the pharmaceutical industry. In some embodiments, the term“consisting essentially of” refers to a composition, whose only activeingredient is the indicated active ingredient, however, other compoundsmay be included which are for stabilizing, preserving, etc. theformulation, but are not involved directly in the therapeutic effect ofthe indicated active ingredient. In some embodiments, the term“consisting essentially of” may refer to components which facilitate therelease of the active ingredient. In some embodiments, the term“consisting” refers to a composition, which contains the activeingredient and a pharmaceutically acceptable carrier or excipient.

Further, as used herein, the term “comprising” is intended to mean thatthe system includes the recited elements, but not excluding others whichmay be optional. By the phrase “consisting essentially of” it is meant amethod that includes the recited elements but exclude other elementsthat may have an essential significant effect on the performance of themethod. “Consisting of” shall thus mean excluding more than traces ofother elements. Embodiments defined by each of these transition termsare within the scope of this invention.

Similarly, in some embodiments, the vector of and for use in the methodsof the present invention comprise a nucleic acid sequence operablylinked to one or more regulatory sequences, wherein said nucleic acidsequence encodes a therapeutic polypeptide. In another embodiment, thevector consists essentially of such a nucleic acid sequence, and inanother embodiment, the vector consists of such a nucleic acid sequence.In one embodiment, the nucleic acid operably linked to one or moreregulatory sequences comprises the nucleic acids of SEQ ID No. 11.

Examples of mammals from which the micro-organs can be isolated includehumans and other primates, swine, such as wholly or partially inbredswine (e.g., miniature swine, and transgenic swine), rodents, etc.Micro-organs may be processed from tissue from a variety of organs,which in one embodiment is the skin, the dermis, the lymph system, thepancreas, the liver, the gallbladder, the kidney, the digestive tract,the respiratory tract, the reproductive system, the urinary tract, theblood, the bladder, the cornea, the prostate, the bone marrow, thethymus, the spleen, or a combination thereof. Explants from these organsmay comprise islet of Langerhans cells, hair follicles, glands,epithelial and connective tissue cells, or a combination thereofarranged in a microarchitecture similar to the microarchitecture of theorgan from which the explant was obtained. In one embodiment, themicroarchitecture of the organ from which the explant was obtained maybe discerned or identified in the micro-organ explant using materials,apparati, and/or methods known in the art.

In one embodiment, the present invention provides a formulation andmethods of use thereof comprising a genetically modified micro-organ. Inone embodiment, the term “genetically modified micro-organ” or “GMMO”refers to a micro-organ that expresses at least one recombinant geneproduct. In other embodiments, reference to a micro-organ does notnecessarily refer to a non-genetically modified micro-organ, but mayalso refer in some instances to a genetically modified micro-organ aswill be clear from the context to one of skill in the art. In oneembodiment, the phrase “gene product” refers to proteins, polypeptides,peptides and functional RNA molecules. In one embodiment, the geneproduct encoded by the nucleic acid molecule is the desired gene productto be supplied to a subject. Examples of such gene products includeproteins, peptides, glycoproteins and lipoproteins normally produced bycells of the recipient subject. In one embodiment, the gene product isnot naturally occurring in the organism from which the micro-organ washarvested and/or in the organism in which the GMMO is implanted, whilein another embodiment, the gene product is naturally occurring. In oneembodiment, the gene product of the GMMO is similar or identical to agene product endogenously expressed by one or more cells of themicro-organ. In one embodiment, genetic modification increases the levelof a gene product that would be produced in a non-genetically modifiedmicro-organ. In another embodiment, the gene product expressed by theGMMO is not similar or identical to a gene product endogenouslyexpressed by one or more cells of the micro-organ. In anotherembodiment, the gene product encoded by the nucleic acid moleculeencodes a molecule that directly or indirectly controls expression of agene of interest. In another embodiment, the gene product encoded by thenucleic acid molecule up-regulates or down-regulates the expressionlevels of the desired gene product to be supplied to a subject.

In another embodiment, genetic modification of a micro-organ may modifythe expression profile of an endogenous gene. This may be achieved, forexample, by introducing an enhancer, or a repressible or inducibleregulatory element for controlling the expression of an endogenous gene.

Any methodology known in the art can be used for genetically alteringthe micro-organ explant. Any one of a number of different vectors can beused, such as viral vectors, plasmid vectors, linear DNA, etc., as knownin the art, to introduce an exogenous nucleic acid fragment encoding atherapeutic agent into target cells and/or tissue. These vectors can beinserted, for example, using infection, transduction, transfection,calcium-phosphate mediated transfection, DEAE-dextran mediatedtransfection, electroporation, liposome-mediated transfection, biolisticgene delivery, liposomal gene delivery using fusogenic and anionicliposomes (which are an alternative to the use of cationic liposomes),direct injection, receptor-mediated uptake, magnetoporation, ultrasound,or any combination thereof, as well as other techniques known in the art(for further detail see, for example, “Methods in Enzymology” Vol.1-317, Academic Press, Current Protocols in Molecular Biology, AusubelF. M. et al. (eds.) Greene Publishing Associates, (1989) and inMolecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al.Cold Spring Harbor Laboratory Press, (1989), or other standardlaboratory manuals). The polynucleotide segments encoding sequences ofinterest can be ligated into an expression vector system suitable fortransducing mammalian cells and for directing the expression ofrecombinant products within the transduced cells. The introduction ofthe exogenous nucleic acid fragment is accomplished by introducing thevector into the vicinity of the micro-organ. Once the exogenous nucleicacid fragment has been incorporated into the cells using any of thetechniques described above or known in the art, the production and/orthe secretion rate of the therapeutic agent encoded by the nucleic acidfragment can be quantified. In one embodiment, the term “exogenous”refers to a substance that originated outside, for example a nucleicacid that originated outside of a cell or tissue.

In one embodiment, a micro-organ of the formulation and methods of thepresent invention comprises a vector, which in one embodiment,facilitates recombinant gene expression. In one embodiment, the vectoris a non-immunogenic gene transfer agent such as a nonviral vector (e.g.DNA plasmids or minicircle DNA), a “gutless” viral vector i.e. withoutendogenous genes (which in one embodiment, is due to a deletion, whilein another embodiment, due to an insertion, substitution or deletion ina gene that prevents gene expression), a helper-dependent adenovirus(HDAd) vector, or adeno associated virus AAV (which in one embodiment issingle stranded and in another embodiment, double stranded). In anotherembodiment, said formulation is so chosen such that recombinant geneexpression results in lack of toxicity or immune-mediated rejection ofthe gene product by the micro-organ. In one embodiment, the vector isvirally derived, and in another embodiment, the vector is a plasmid. Inone embodiment, the virally-derived vector is derived from adenovirus,which in one embodiment, is helper-dependent adenovirus, while inanother embodiment, the virally-derived vector is derived fromadenovirus-associated vector, as is described herein below.

In one embodiment, the term “vector” or “expression vector” refers to acarrier molecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. In one embodiment,the nucleic acid molecules are transcribed into RNA, which in some casesare then translated into a protein, polypeptide, or peptide. In othercases, RNA sequences are not translated, for example, in the productionof antisense molecules or ribozymes. In one embodiment, expressionvectors can contain a variety of “control sequences” which refer tonucleic acid sequences necessary for the transcription and possiblytranslation of an operably linked coding sequence in a particular hostcell. In another embodiment, a vector further includes an origin ofreplication. As used herein, the term “control sequence” may also bereferred to herein as a “regulatory sequence”. In one embodiment thevector may be a shuttle vector, which in one embodiment can propagateboth in prokaryotic and eukaryotic cells, or in another embodiment, thevector may be constructed to facilitate its integration within thegenome of an organism of choice. The vector, in other embodiments maybe, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, avirus or an artificial chromosome. In one embodiment, the vector is aviral vector, which in one embodiment may be a bacteriophage, mammalianvirus, or plant virus.

In one embodiment, the viral vector is an adenoviral vector. In anotherembodiment, the adenovirus may be of any known serotype or subgroup.

Advantages of using an adenoviral vector as a gene transfer vector are:its mid-sized genome, ease of manipulation, high titer, wide target-cellrange and high infectivity. Both ends of the adenoviral genome contain100-200 base pair inverted repeats (ITRs), which are cis elementsnecessary for viral DNA replication and packaging. The early (E) andlate (L) regions of the genome contain different transcription unitsthat are divided by the onset of viral DNA replication. The E1 region(E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off. Theproducts of the late genes, including the majority of the viral capsidproteins, are expressed only after significant processing of a singleprimary transcript issued by the major late promoter (MLP). The MLP,(located at 16.8 m.u.) is particularly efficient during the late phaseof infection, and all the mRNAs issued from this promoter possess a5′-tripartite leader (TPL) sequence which makes them preferred mRNAs fortranslation.

In another embodiment, the adenoviral vector is a helper-dependentadenoviral vector (“HDAD”, “HD” or “HDAd” or “HD-Ad”), which in anotherembodiment, is synonymous with gutless, gutted, mini, fully deleted,high-capacity, Δ, or pseudo adenovirus, and which in another embodimentare deleted of all viral coding sequences except for sequencessupporting DNA replication, which in one embodiment, comprise theadenovirus inverted terminal repeats and packaging sequence (ψ). Inanother embodiment, helper-dependent adenoviruses express no viralproteins. In one embodiment, a helper-dependent adenoviral vectorcomprises only the cis-acting elements of the adenovirus required toreplicate and package the vector DNA. In one embodiment, ahelper-dependent adenoviral vector comprises approximately 500 bp ofwild-type adenovirus sequence. In another embodiment, the adenoviralvector additionally comprises stuffer DNA to meet the minimumrequirement for a genome size of 27.7 kb, which in one embodiment isrequired for efficient packaging into the adenovirus capsid. In oneembodiment, non-coding mammalian DNA, with minimal repeat sequences, isused as stuffer DNA. In another embodiment, stuffer DNA comprisesnon-mammalian DNA, which in one embodiment, is HPRT and/or C346 cosmidsequences. In one embodiment, the HDAd vector is a non-replicatingvector.

In one embodiment, helper-dependent adenoviruses display high-efficiencyin vivo transduction, high-level transgene expression, are able tomaintain long-term transgene expression, in one embodiment, by avoidingchronic toxicity due to residual expression of viral proteins, or acombination thereof. In another embodiment, helper-dependentadenoviruses have high titer production, efficient infection of a broadrange of cell types, the ability to infect dividing and nondividingcells, or a combination thereof. In yet another embodiment, ahelper-dependent adenovirus for use in the methods of the instantinvention does not induce a strong adaptive immune response to animplanted micro-organ, which in one embodiment, is characterized by thegeneration of adeno-specific MHC class I restricted CD8 cytotoxic Tlymphocytes (CTL) in immunocompetent hosts, which in one embodiment,would limit the duration of transgene expression and in anotherembodiment, would result in adenovirus vector clearance within severalweeks. In still another embodiment, a helper-dependent adenovirus foruse in the methods of the instant invention does not induce highcytotoxic T cell levels (as may be measured in one embodiment bypositive CD8 staining, as is known in the art), and, in anotherembodiment, does not induce high helper T cell levels (as may bemeasured in one embodiment by positive CD4 stain, as is known in theart).

In another embodiment, helper-dependent adenoviruses have a lower riskof germ line transmission and insertional mutagenesis that may causeoncogenic transformation, because the vector genome does not integrateinto the host cell chromosomes. In one embodiment, the cloning capacityof helper-dependent adenoviruses is very large (in one embodiment,approximately 37 kb, in another embodiment, approximately 36 kb),allowing for the delivery of whole genomic loci, multiple transgenes,and large cis-acting elements to enhance, prolong, and regulatetransgene expression.

In one embodiment, the helper-dependent adenovirus system for use withthe compositions and in the methods of the present invention is similarto that described in Palmer and Ng, 2003 (Mol Ther 8:846) and in Palmerand Ng, 2004 (Mol Ther 10:792), which are hereby incorporated herein byreference in their entirety. In one embodiment, there is a stuffersequence inserted into the E3 region of the helper virus component ofthe helper-dependent adenovirus system to minimize recombination betweenthe helper adenovirus and the helper-dependent adenovirus to producereplication competent adenovirus.

In one embodiment, formulations of the instant invention comprisinghelper-dependent adenoviral vectors demonstrate long-term, high in vitro(FIGS. 1, 2, and 6B) and in vivo (FIG. 6A) expression levels of EPO andIFN-alpha. In another embodiment, formulations of the instant inventioncomprising helper-dependent adenoviral vectors demonstrate an increasedpercent of peak EPO expression levels for at least 100 dayspost-transduction compared to micro-organs comprising adenovirus-5 (FIG.3). Without being bound by theory, one factor that may contribute to thelong-lasting, high levels of gene product from micro-organs of theinstant invention is use of a helper-dependent adenovirus vector, whichis non-toxic to tissue and non-immunogenic within the formulations ofthe present invention.

In yet another embodiment, formulations of the instant inventioncomprising helper-dependent adenoviral vectors demonstrate an increasedpercent of peak EPO expression levels in vivo for less than one month(FIG. 16). In still another embodiment, in vivo peak EPO expressionlevels are increased for less than two weeks (FIG. 10C).

In another embodiment, the adenoviral vector is E1-deleted, while inanother embodiment, the adenoviral vector additionally comprisesdeletions for E2, E3, E4, or a combination thereof.

In another embodiment, the viral vector is an adeno-associated viralvector (AAV). In one embodiment, AAV is a parvovirus, discovered as acontamination of adenoviral stocks. It is a ubiquitous virus (antibodiesare present in 85% of the US human population) that has not been linkedto any disease. It is also classified as a dependovirus, because itsreplication is dependent on the presence of a helper virus, such asadenovirus. At least nine serotypes have been isolated, of which AAV-2is the best characterized. AAV has a single-stranded linear DNA that isencapsidated into capsid proteins VP1, VP2 and VP3 to form anicosahedral virion of 20 to 24 nm in diameter.

In one embodiment, the AAV DNA is approximately 4.7 kilobases long. Inone embodiment, it contains two open reading frames and is flanked bytwo ITRs. There are two major genes in the AAV genome: rep and cap. Therep gene codes for proteins responsible for viral replications, whereascap codes for capsid protein VP1-3. Each ITR forms a T-shaped hairpinstructure. These terminal repeats are the only essential cis componentsof the AAV for chromosomal integration. Therefore, in one embodiment,the AAV can be used as a vector with all viral coding sequences removedand replaced by the cassette of genes for delivery.

In one embodiment, when using recombinant AAV (rAAV) as an expressionvector, the vector comprises the 145-bp ITRs, which are only 6% of theAAV genome, which in one embodiment, leaves space in the vector toassemble a 4.5-kb DNA insertion.

In one embodiment, AAV is safe in that it is not considered pathogenicnor is it associated with any disease. The removal of viral codingsequences minimizes immune reactions to viral gene expression, andtherefore, rAAV evokes only a minimal inflammatory response, if any. Inanother embodiment, AAV vector is double-stranded, while in anotherembodiment, AAV vector is self-complementary, which in one embodiment,bypasses the requirement of viral second-strand DNA synthesis, which inone embodiment, results in early transgene expression.

In another embodiment, the viral vector is a retroviral vector. Theretroviruses are a group of single-stranded RNA viruses characterized byan ability to convert their RNA to double-stranded DNA in infected cellsby a process of reverse-transcription. The resulting DNA then stablyintegrates into cellular chromosomes as a provirus and directs synthesisof viral proteins. The integration results in the retention of the viralgene sequences in the recipient cell and its descendants. The retroviralgenome contains three genes, gag, pol, and env that code for capsidproteins, polymerase enzyme, and envelope components, respectively. Asequence found upstream from the gag gene contains a signal forpackaging of the genome into virions. Two long terminal repeat (LTR)sequences are present at the 5′ and 3′ ends of the viral genome. Thesecontain strong promoter and enhancer sequences and are also required forintegration in the host cell genome.

In order to construct a retroviral vector in one embodiment, a nucleicacid encoding one or more oligonucleotide or polynucleotide sequences ofinterest is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol, and envgenes but without the LTR and packaging components is constructed. Whena recombinant plasmid containing a cDNA, together with the retroviralLTR and packaging sequences is introduced into this cell line (bycalcium phosphate precipitation, for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media. Themedia containing the recombinant retroviruses is then collected,optionally concentrated, and used for gene transfer. Retroviral vectorsare able to infect a broad variety of cell types. However, integrationand stable expression require the division of host cells.

In other embodiments, the viral vector is derived from a virus such asvaccinia virus, lentivirus, polio virus, hepatitis virus, papillomavirus, cytomegalovirus, simian virus, or herpes simplex virus.

In certain embodiments of the invention, the vector comprising a nucleicacid sequence may comprise naked recombinant DNA or plasmids. Transferof the construct may be performed by any method which physically orchemically permeabilizes the cell membrane. In one embodiment, thevector is a mini-circle DNA, which in one embodiment, is a supercoiledDNA molecule for non-viral gene transfer, which has neither a bacterialorigin of replication nor an antibiotic resistance marker. In anotherembodiment, mini-circle DNA comprises no bacterial control regions fromgene delivery vectors during the process of plasmid production. They arethus smaller and potentially safer than other plasmids used in genetherapy. In one embodiment, mini-circle DNA produce high yield, aresimple to purify, and provide robust and persistent transgeneexpression.

Construction of vectors using standard recombinant techniques is wellknown in the art (see, for example, Maniatis, et al., Molecular Cloning,A Laboratory Manual (Cold Spring Harbor, 1990) and Ausubel, et al.,1994, Current Protocols in Molecular Biology (John Wiley & Sons, 1996),both incorporated herein by reference).

In another embodiment, a vector further comprises an insertion of aheterologous nucleic acid sequence encoding a marker polypeptide. Themarker polypeptide may comprise, for example, yECitrine, greenfluorescent protein (GFP), DS-Red (red fluorescent protein), secretedalkaline phosphatase (SEAP), β-galactosidase, chloramphenicol acetyltransferase, luciferase, GFP/EGFP, human growth hormone, or any numberof other reporter proteins known to one skilled in the art.

In another embodiment, the vectors may comprise one or more genes ofinterest. Thus, in one embodiment, a vector of the instant invention maycomprise a gene of interest, which in one embodiment, is erythropoietinor interferon alpha2b, which in one embodiment, expresses a marker, andin another embodiment, is linked in frame to a marker, which in oneembodiment allows identification of the gene product of interest and inanother embodiment, allows the distinction between a gene product ofinterest produced by a micro-organ and a similar gene product producedendogenously by host cells outside of the micro-organ(s).

In one embodiment, a vector comprising a nucleic acid encoding atherapeutic polypeptide of the instant invention is introduced into amicro-organ. There are a number of techniques known in the art forintroducing cassettes and/or vectors into cells, for affecting themethods of the present invention, such as, but not limited to: directDNA uptake techniques, and virus, plasmid, linear DNA or liposomemediated transduction, receptor-mediated uptake and magnetoporationmethods employing calcium-phosphate mediated and DEAE-dextran mediatedmethods of introduction, electroporation or liposome-mediatedtransfection, (for further detail see, for example, “Methods inEnzymology” Vol. 1-317, Academic Press, Current Protocols in MolecularBiology, Ausubel F. M. et al. (eds.) Greene Publishing Associates,(1989) and in Molecular Cloning: A Laboratory Manual, 2nd Edition,Sambrook et al. Cold Spring Harbor Laboratory Press, (1989), or otherstandard laboratory manuals).

In one embodiment, bombardment with nucleic acid coated particles may bea method for transferring a naked DNA expression construct into cells.This method depends on the ability to accelerate DNA-coatedmicro-projectiles to a high velocity allowing them to pierce cellmembranes and enter cells without killing them. Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force. The micro-projectiles used havecomprised biologically inert or biocompatible substances such astungsten or gold beads. It is to be understood that any of these methodsmay be utilized for introduction of the desired sequences into cells,and cells thereby produced are to be considered as part of thisinvention, as is their use for effecting the methods of this invention.

In one embodiment, the vectors of the formulations and methods of theinstant invention comprise a nucleic acid sequence. As used herein, theterm “nucleic acid” refers to polynucleotide or to oligonucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA) or mimetic thereof. The term should also be understood to include,as equivalents, analogs of RNA or DNA made from nucleotide analogs, and,as applicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotide. This term includesoligonucleotides composed of naturally occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

In one embodiment, the term “nucleic acid” or “oligonucleotide” refersto a molecule, which may include, but is not limited to, prokaryoticsequences, eukaryotic mRNA, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. The term also refers to sequences that include any of theknown base analogs of DNA and RNA.

The nucleic acids can be produced by any synthetic or recombinantprocess, which are well known in the art. Nucleic acids can further bemodified to alter biophysical or biological properties by means oftechniques known in the art. For example, the nucleic acid can bemodified to increase its stability against nucleases (e.g.,“end-capping”), or to modify its solubility, or binding affinity tocomplementary sequences. These nucleic acids may comprise the vector,the expression cassette, the promoter sequence, the gene of interest, orany combination thereof. In another embodiment, its lipophilicity may bemodified, which, in turn, will reflect changes in the systems employedfor its delivery, and in one embodiment, may further be influenced bywhether such sequences are desired for retention within, or permeationthrough the skin, or any of its layers. Such considerations mayinfluence any compound used in this invention, in the methods andsystems described.

In one embodiment, DNA can be synthesized chemically from the fournucleotides in whole or in part by methods known in the art. Suchmethods include those described in Caruthers (1985; Science230:281-285). DNA can also be synthesized by preparing overlappingdouble-stranded oligonucleotides, filling in the gaps, and ligating theends together (see, generally, Sambrook et al. (1989; MolecularCloning—A Laboratory Manual, 2nd Edition. Cold Spring Habour LaboratoryPress, New York)). In another embodiment, inactivating mutations may beprepared from wild-type DNA by site-directed mutagenesis (see, forexample, Zoller et al. (1982; DNA. 1984 December; 3(6):479-88); Zoller(1983); and Zoller (1984; DNA. 1984 December; 3(6):479-88); McPherson(1991; Directed Mutagenesis: A Practical Approach. Oxford UniversityPress, NY)). The DNA obtained can be amplified by methods known in theart. One suitable method is the polymerase chain reaction (PCR) methoddescribed in Saiki et al. (1988; Science. 1988 Jan. 29;239(4839):487-491), Mullis et al., U.S. Pat. No. 4,683,195, and Sambrooket al. (1989).

Methods for modifying nucleic acids to achieve specific purposes aredisclosed in the art, for example, in Sambrook et al. (1989). Moreover,the nucleic acid sequences of the invention can include one or moreportions of nucleotide sequence that are non-coding for the protein ofinterest. Variations in DNA sequences, which are caused by pointmutations or by induced modifications (including insertion, deletion,and substitution) to enhance the activity, half-life or production ofthe polypeptides encoded thereby, are also encompassed in the invention.

The formulations of this invention may comprise nucleic acids, in oneembodiment, or in another embodiment, the methods of this invention mayinclude delivery of the same, wherein, in another embodiment, thenucleic acid is a part of a vector.

The efficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art as described hereinbelow.

As will be appreciated by one skilled in the art, a fragment orderivative of a nucleic acid sequence or gene that encodes for a proteinor peptide can still function in the same manner as the entire wild typegene or sequence. Likewise, forms of nucleic acid sequences can havevariations as compared to wild type sequences, nevertheless encoding theprotein or peptide of interest, or fragments thereof, retaining wildtype function exhibiting the same biological effect, despite thesevariations. Each of these represents a separate embodiment of thispresent invention.

In one embodiment, the formulations and methods of the present inventionmay be used for gene silencing applications. In one embodiment, theactivity or function of a particular gene is suppressed or diminished,via the use of anti-sense oligonucleotides, which are chimericmolecules, containing two or more chemically distinct regions, each madeup of at least one nucleotide.

In one embodiment, chimeric oligonucleotides comprise at least oneregion wherein the oligonucleotide is modified so as to confer upon theoligonucleotide an increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget polynucleotide. An additional region of the oligonucleotide mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids, which according to this aspect of the invention, serves as ameans of gene silencing via degradation of specific sequences. Cleavageof the RNA target can be routinely detected by gel electrophoresis and,if necessary, associated nucleic acid hybridization techniques known inthe art.

The chimeric antisense oligonucleotides may, in one embodiment, beformed as composite structures of two or more oligonucleotides and/ormodified oligonucleotides, as is described in the art (see, for example,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922), and may, in another embodiment, comprise a ribozymesequence.

Inhibition of gene expression, activity or function is effected, inanother embodiment, via the use of small interfering RNAs, whichprovides sequence-specific inhibition of gene expression. Administrationof double stranded/duplex RNA (dsRNA) corresponding to a single gene inan organism can silence expression of the specific gene by rapiddegradation of the mRNA in affected cells. This process is referred toas gene silencing, with the dsRNA functioning as a specific RNAinhibitor (RNAi). RNAi may be derived from natural sources, such as inendogenous virus and transposon activity, or it can be artificiallyintroduced into cells (Elbashir S M, et al (2001). Nature 411:494-498)via microinjection (Fire et al. (1998) Nature 391: 806-11), or bytransformation with gene constructs generating complementary RNAs orfold-back RNA, or by other vectors (Waterhouse, P. M., et al. (1998).Proc. Natl. Acad. Sci. USA 95, 13959-13964 and Wang, Z., et al. (2000).J. Biol. Chem. 275, 40174-40179). The RNAi mediating mRNA degradation,in one embodiment, comprises duplex or double-stranded RNA, or, in otherembodiments, include single-stranded RNA, isolated RNA (partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA), as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion and/or alteration of one or morenucleotides.

In one embodiment, the nucleic acid of the formulations and methods ofthe instant invention encode a therapeutic polypeptide. In oneembodiment, the term “polypeptide” refers to a molecule comprised ofamino acid residues joined by peptide (i.e., amide) bonds and includespeptides, polypeptides, and proteins. Hence, in one embodiment, thepolypeptides of this invention may have single or multiple chains ofcovalently linked amino acids and may further contain intrachain orinterchain linkages comprised of disulfide bonds. In one embodiment,some polypeptides may also form a subunit of a multiunit macromolecularcomplex. In one embodiment, the polypeptides can be expected to possessconformational preferences and to exhibit a three-dimensional structure.Both the conformational preferences and the three-dimensional structurewill usually be defined by the polypeptide's primary (i.e., amino acid)sequence and/or the presence (or absence) of disulfide bonds or othercovalent or non-covalent intrachain or interchain interactions.

In one embodiment, the term “peptide” refers to native peptides (eitherdegradation products, synthetically synthesized peptides or recombinantpeptides) and/or peptidomimetics (typically, synthetically synthesizedpeptides), such as peptoids and semipeptoids which are peptide analogs,which may have, for example, modifications rendering the peptides morestable while in a body or more capable of penetrating into cells. Suchmodifications include, but are not limited to N terminus modification, Cterminus modification, peptide bond modification, including, but notlimited to, CH₂—NH, CH₂—S, CH₂—S═O, O=C—NH, CH₂—O, CH₂—CH₂, S═C—NH,CH═CH or CF═CH, backbone modifications, and residue modification.Methods for preparing peptidomimetic compounds are well known in the artand are specified, for example, in Quantitative Drug Design, C. A.Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which isincorporated by reference as if fully set forth herein.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH₃)—CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH₂—), aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH₂—NH—), hydroxyethylene bonds (—CH(OH)—CH₂—), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH₂—CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom. These modifications canoccur at any of the bonds along the peptide chain and even at several(2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as TIC, naphthylelanine (Nol),ring-methylated derivatives of Phe, halogenated derivatives of Phe oro-methyl-Tyr. In addition to the above, the peptides of the presentinvention may also include one or more modified amino acids or one ormore non-amino acid monomers (e.g. fatty acids, complex carbohydratesetc).

In one embodiment, the term “amino acid” or “amino acids” is understoodto include the 20 naturally occurring amino acids; those amino acidsoften modified post-translationally in vivo, including, for example,hydroxyproline, phosphoserine and phosphothreonine; and other unusualamino acids including, but not limited to, 2-aminoadipic acid,hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.Furthermore, the term “amino acid” may include both D- and L-aminoacids.

As used herein, the term “amino acid” refers to either the D or Lstereoisomer form of the amino acid, unless otherwise specificallydesignated. Also encompassed within the scope of this invention areequivalent proteins or equivalent peptides, e.g., having the biologicalactivity of purified wild type tumor suppressor protein. “Equivalentproteins” and “equivalent polypeptides” refer to compounds that departfrom the linear sequence of the naturally occurring proteins orpolypeptides, but which have amino acid substitutions that do not changeit's biologically activity. These equivalents can differ from the nativesequences by the replacement of one or more amino acids with relatedamino acids, for example, similarly charged amino acids, or thesubstitution or modification of side chains or functional groups.

The peptides or polypeptides, or the DNA sequences encoding same, may beobtained from a variety of natural or unnatural sources, such as aprokaryotic or a eukaryotic cell. In one embodiment, the source cell maybe wild type, recombinant, or mutant. In another embodiment, theplurality of peptides or polypeptides may be endogenous tomicroorganisms, such as bacteria, yeast, or fungi, to a virus, to ananimal (including mammals, invertebrates, reptiles, birds, and insects)or to a plant cell.

In another embodiment, the peptides or polypeptides may be obtained frommore specific sources, such as the surface coat of a virion particle, aparticular cell lysate, a tissue extract, or they may be restricted tothose polypeptides that are expressed on the surface of a cell membrane.

In another embodiment, the peptide or polypeptide is derived from aparticular cell or tissue type, developmental stage or disease conditionor stage. In one embodiment, the disease condition or stage is cancer,in another embodiment, the disease condition is an infection, which inanother embodiment, is an HIV infection. In another embodiment, thedisease condition is a developmental disorder, while in anotherembodiment, the disease condition is a metabolic disorder.

The polypeptide of the present invention can be of any size. As can beexpected, the polypeptides can exhibit a wide variety of molecularweights, some exceeding 150 to 200 kilodaltons (kD). Typically, thepolypeptides may have a molecular weight ranging from about 5,000 toabout 100,000 daltons. Still others may fall in a narrower range, forexample, about 10,000 to about 75,000 daltons, or about 20,000 to about50,000 daltons. In an alternative embodiment, the polypeptides of thepresent invention may be 1-250 amino acid residues long. In anotherembodiment, the polypeptides of the present invention may be 10-200amino acid residues long. In an alternative embodiment, the polypeptidesof the present invention may be 50-100 amino acid residues long. In analternative embodiment, the polypeptides of the present invention may be1-250 amino acid residues long. In an alternative embodiment, thepolypeptides of the present invention may be 1-250 amino acid residueslong. In one embodiment, the maximum size of the peptide or polypeptideis determined by the vector from which it is expressed, which in oneembodiment, is approximately between 20 and 37 kD, between 20 and 25 kD,between 25 and 30 kD, between 30 and 37 kD, or between 35 and 37 kD. Inanother embodiment, the polypeptide is a 34 kD glycoprotein.

In another embodiment, the peptides or polypeptides are agonists. Inanother embodiment, the peptides or polypeptides are antagonists. Inanother embodiment, the peptides or polypeptides are antigens. Inanother embodiment, the peptides or polypeptides are enzymes. In anotherembodiment, the peptides or polypeptides are activators of enzymes orother substrates. In another embodiment, the peptides or polypeptidesare inhibitors of enzymes or other substrates. In another embodiment,the peptides or polypeptides are hormones. In another embodiment, thepeptides or polypeptides are regulatory proteins. Regulatory proteinscommand the numerous interactions that govern the expression andreplication of genes, the performance of enzymes, the interplay betweencells and their environment, and many other manifestations. In anotherembodiment, the peptides or polypeptides are cytoskeletal proteins.Cytoskeletal proteins form a flexible framework for the cell, provideattachment points for organelles and formed bodies, and makecommunication between parts of the cell possible. In another embodiment,the peptides or polypeptides are toxins. In another embodiment, thetherapeutic nucleic acids of the present invention encode one or moresuicide genes.

In another embodiment, the peptides or polypeptides are functionalfragments of agonists, antagonists, antigens, enzymes, enzymeactivators, enzyme inhibitors, enzyme substrates, hormones, regulatoryproteins, cytoskeletal proteins, or toxins. “Functional fragments” aremeant to indicate a portion of the peptide or polypeptide which iscapable of performing one or more of the functions of the peptide orpolypeptide, even in the absence of the remainder of the peptide orpolypeptide. In one embodiment, the functional fragment is sufficient tomediate an intermolecular interaction with a target of interest.

In an alternative embodiment, the peptide binds DNA or RNA or a fragmentthereof. In one embodiment, the DNA or RNA binding peptide may be any ofthe many known in the art including, but not limited to Zinc fingerproteins such as Beta-beta-alpha zinc finger proteins, Nuclear receptorproteins, Loop-sheet-helix type protein, and GAL4 type protein; theHelix-turn-helix proteins such as Cro and repressor proteins, LacIpurine repressor proteins (PurR), Fold restriction endonuclease(DNA-recognition region), Gamma-delta recombinase protein (C-terminaldomain), Hin recombinase protein, Trp repressor protein, Diptheria toxrepressor, Catabolite gene activator proteins (CAP), Homeodomainproteins, RAP1 protein, Prd paired protein, Tc3 transposase protein,TFIIB family, Interferon regulatory factor, Transcription factor family,and ETS domain family bacteriophage; and the Leucine zipper proteinssuch as Basic zipper proteins and Zipper-type proteins(helix-loop-helix). In another embodiment, the DNA or RNA bindingpeptide may be other alpha-helix proteins such as Cre recombinasefamily, Papillomavirus-1 E2 protein, Histone family, Ebna1 nuclearprotein family, Skn-1 transcription factor, High mobility group family,and MADS box family; Beta-sheet proteins such as TATA Box-BindingProteins; Beta-hairpin/ribbon proteins such as Met repressor protein,Tus replication terminator protein, Integration host factor protein,Hyperthermophile DNA binding protein, Arc repressor, Transcriptionfactor T domain; and other protein families such as Rel homology regionproteins and Stat family. In another embodiment, the DNA or RNA bindingpeptide may be enzymes such as Methyl transferase proteins, PvuIIEndonuclease protein, Endonuclease V protein, EcoRV Endonuclease family,BamHI Endonuclease family, EcoRI endonuclease family, DNA mismatchendonuclease, DNA polymerase I protein, DNA polymerase T7, Dnase Iproteins, DNA polymerase beta proteins, Uraci-DNA glycosylase,Methyladenine-DNA glycosylase, Homing endonuclease, and Topoisomerase Ior viral proteins such as HIV reverse transcriptase.

In another embodiment, the peptide or polypeptide is a transcriptionalor translational activator or a fragment thereof. In another embodiment,the peptide or polypeptide is a transcriptional or translationalrepressor or a fragment thereof. In another embodiment, the peptide orpolypeptide is a receptor or a fragment thereof.

In one embodiment, the peptide or polypeptide may represent a cognatepeptide of any of the peptides or polypeptides described hereinabove. A“cognate” peptide is any peptide that interacts and/or binds to anothermolecule.

According to other embodiments of the present invention, recombinantgene products may be encoded by a polynucleotide having a modifiednucleotide sequence, as compared to a corresponding naturalpolynucleotide.

In addition to proteins, recombinant gene products may also comprisefunctional RNA molecules.

According to another embodiment of the present invention, theformulations and methods of the present invention may provide amicro-organ producing functional RNA molecules. Functional RNA moleculesmay comprise antisense oligonucleotide sequences, ribozymes comprisingthe antisense oligonucleotide described herein and a ribozyme sequencefused thereto. Such a ribozyme is readily synthesizable using solidphase oligonucleotide synthesis.

Ribozymes are being increasingly used for the sequence-specificinhibition of gene expression by the cleavage of mRNAs encoding proteinsof interest [Welch et al., “Expression of ribozymes in gene transfersystems to modulate target RNA levels.” Curr Opin Biotechnol. 1998October; 9(5):486-96]. The possibility of designing ribozymes to cleaveany specific target RNA has rendered them valuable tools in both basicresearch and therapeutic applications. In the therapeutics area,ribozymes have been exploited to target viral RNAs in infectiousdiseases, dominant oncogenes in cancers and specific somatic mutationsin genetic disorders [Welch et al., “Ribozyme gene therapy for hepatitisC virus infection.” Clin Diagn Virol. Jul. 15, 1998; 10(2-3):163-71.Most notably, several ribozyme gene therapy protocols for HIV patientsare already in Phase 1 trials. More recently, ribozymes have been usedfor transgenic animal research, gene target validation and pathwayelucidation. Several ribozymes are in various stages of clinical trials.ANGIOZYME was the first chemically synthesized ribozyme to be studied inhuman clinical trials. ANGIOZYME specifically inhibits formation of theVEGF-r (Vascular Endothelial Growth Factor receptor), a key component inthe angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well asother firms has demonstrated the importance of anti-angiogenesistherapeutics in animal models. HEPTAZYME, a ribozyme designed toselectively destroy Hepatitis C Virus (HCV) RNA, was found effective indecreasing Hepatitis C viral RNA in cell culture assays.

As described hereinabove, in one embodiment, the formulations andmethods of the present invention provide a therapeutic formulationcomprising a nucleic acid sequence encoding a therapeutic polypeptide.In one embodiment, the term “therapeutic” refers to a molecule, whichwhen provided to a subject in need, provides a beneficial effect. Insome cases, the molecule is therapeutic in that it functions to replacean absence or diminished presence of such a molecule in a subject. Inone embodiment, the therapeutic protein is that of a protein which isabsent in a subject, such as in cases of subjects with an endogenousnull or mis-sense mutation of a required protein. In other embodiments,the endogenous protein is mutated, and produces a non-functionalprotein, compensated for by the provision of the functional protein. Inother embodiments, expression of a heterologous protein is additive tolow endogenous levels, resulting in cumulative enhanced expression of agiven protein. In other embodiments, the molecule stimulates a signalingcascade that provides for expression, or secretion, or others of acritical element for cellular or host functioning.

In one embodiment, the term “therapeutic formulation” describes asubstance applicable for use in the diagnosis, or in another embodiment,cure, or in another embodiment, mitigation, or in another embodiment,treatment, or in another embodiment, prevention of a disease, disorder,condition or infection. In one embodiment, the “therapeutic formulation”of this invention refers to any substance which affect the structure orfunction of the target to which it is applied.

In another embodiment, the “therapeutic formulation” of the presentinvention is a molecule that alleviates a symptom of a disease ordisorder when administered to a subject afflicted thereof. In oneembodiment, the “therapeutic formulation” of this invention is asynthetic molecule, or in another embodiment, a naturally occurringcompound isolated from a source found in nature.

In one embodiment, the therapeutic polypeptide is erythropoietin. In oneembodiment, the beneficial effect provided by erythropoietin isincreased Hb levels. In one embodiment, the beneficial effect providedby erythropoietin is treatment of anemia.

In another embodiment, the therapeutic polypeptide is interferon alpha,which in one embodiment, is interferon alpha 2b. In one embodiment, saidtherapeutic polypeptide is any other therapeutic polypeptide.

In one embodiment, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor lessen the targeted pathologic condition or disorder as describedhereinabove. Thus, in one embodiment, treating may include directlyaffecting or curing, suppressing, inhibiting, preventing, reducing theseverity of, delaying the onset of, reducing symptoms associated withthe disease, disorder or condition, or a combination thereof. Thus, inone embodiment, “treating” refers inter alia to delaying progression,expediting remission, inducing remission, augmenting remission, speedingrecovery, increasing efficacy of or decreasing resistance to alternativetherapeutics, or a combination thereof. In one embodiment, “preventing”refers, inter alia, to delaying the onset of symptoms, preventingrelapse to a disease, decreasing the number or frequency of relapseepisodes, increasing latency between symptomatic episodes, or acombination thereof. In one embodiment, “suppressing” or “inhibiting”,refers inter alia to reducing the severity of symptoms, reducing theseverity of an acute episode, reducing the number of symptoms, reducingthe incidence of disease-related symptoms, reducing the latency ofsymptoms, ameliorating symptoms, reducing secondary symptoms, reducingsecondary infections, prolonging patient survival, or a combinationthereof.

In one embodiment, symptoms are primary, while in another embodiment,symptoms are secondary. In one embodiment, “primary” refers to a symptomthat is a direct result of a particular disease, while in oneembodiment; “secondary” refers to a symptom that is derived from orconsequent to a primary cause. In one embodiment, the compounds for usein the present invention treat primary or secondary symptoms orsecondary complications related to the disease. In another embodiment,“symptoms” may be any manifestation of a disease or pathologicalcondition.

In one embodiment, a therapeutic nucleic acid may encode a therapeuticpolypeptide, which may in one embodiment, comprise an enzyme, an enzymecofactor, a cytotoxic protein, an antibody, a channel protein, atransporter protein, a growth factor, a hormone, a cytokine, a receptor,a mucin, a surfactant, an aptamer or a hormone. In another embodiment,the therapeutic polypeptide may be of one or more of the categories asdescribed above. In another embodiment, a therapeutic nucleic acid mayencode functional RNA as described hereinbelow.

In one embodiment, the term “antibody or antibody fragment” refers tointact antibody molecules as well as functional fragments thereof, suchas Fab, F(ab′)2, and Fv that are capable of binding to an epitope. Inone embodiment, an Fab fragment refers to the fragment which contains amonovalent antigen-binding fragment of an antibody molecule, which canbe produced by digestion of whole antibody with the enzyme papain toyield an intact light chain and a portion of one heavy chain. In oneembodiment, Fab′ fragment refers to a part of an antibody molecule thatcan be obtained by treating whole antibody with pepsin, followed byreduction, to yield an intact light chain and a portion of the heavychain. Two Fab′ fragments may be obtained per antibody molecule. In oneembodiment, (Fab′)₂ refers to a fragment of an antibody that can beobtained by treating whole antibody with the enzyme pepsin withoutsubsequent reduction. In another embodiment, F(ab′)₂ is a dimer of twoFab′ fragments held together by two disulfide bonds. In one embodiment,Fv, may refer to a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains. In one embodiment, the antibody fragmentmay be a single chain antibody (“SCA”), a genetically engineeredmolecule containing the variable region of the light chain and thevariable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 1988, incorporated herein by reference).

In one embodiment, the antibody will recognize an epitope, which inanother embodiment, refers to antigenic determinant on an antigen towhich the paratope of an antibody binds. Epitopic determinants may, inother embodiments, consist of chemically active surface groupings ofmolecules such as amino acids or carbohydrate side chains and in otherembodiments, may have specific three dimensional structuralcharacteristics, and/or in other embodiments, have specific chargecharacteristics.

In one embodiment, the epitope recognized is from a pathogen, or inanother embodiment, a pathogenic cell, or in another embodiment, aprotein aberrantly expressed, which, in another embodiment, may refer tothe location, quantity, or combination thereof of expression.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry,Methods, 2: 106-10, 1991.

In one embodiment, the antibody is tumoricidal, and is therebytherapeutic in certain cancers. Antibodies that possess tumoricidalactivity are also known in the art, the use of any of which mayrepresent an embodiment of this invention, including IMC-C225, EMD72000, OvaRex Mab B43.13, anti-ganglioside G(D2) antibody ch14.18,C017-1A, trastuzumab, rhuMAb VEGF, sc-321, AF349, BAF349, AF743, BAF743,MAB743, AB1875, Anti-Flt-4AB3127, FLT41-A, rituximab, 2C3, CAMPATH 1H,2G7, Alpha IR-3, ABX-EGF, MDX-447, anti-p75 IL-2R, anti-p64 IL-2R, and2A11.

In one embodiment, the “therapeutic nucleic acid” of this invention mayencode or the “therapeutic polypeptide” may be molecules that serve asantihypertensives, antidepressants, antianxiety agents, anticlottingagents, anticonvulsants, blood glucose-lowering agents, decongestants,antihistamines, antitussives, anti-inflammatories, antipsychotic agents,cognitive enhancers, cholesterol-reducing agents, antiobesity agents,autoimmune disorder agents, anti-impotence agents, antibacterial andantifungal agents, hypnotic agents, anti-Parkinsonism agents,antibiotics, antiviral agents, anti-neoplastics, barbituates, sedatives,nutritional agents, beta blockers, emetics, anti-emetics, diuretics,anticoagulants, cardiotonics, androgens, corticoids, anabolic agents,growth hormone secretagogues, anti-infective agents, coronaryvasodilators, carbonic anhydrase inhibitors, antiprotozoals,gastrointestinal agents, serotonin antagonists, anesthetics,hypoglycemic agents, dopaminergic agents, anti-Alzheimer's Diseaseagents, anti-ulcer agents, platelet inhibitors and glycogenphosphorylase inhibitors.

In one embodiment, the “therapeutic formulation” of this invention isantibacterial, antiviral, antifungal or antiparasitic. In anotherembodiment, the therapeutic formulation has cytotoxic or anti-canceractivity. In another embodiment, the therapeutic formulation isimmunostimulatory. In another embodiment, the therapeutic formulationinhibits inflammatory or immune responses.

In one embodiment, the therapeutic nucleic acids may encode or thetherapeutic polypeptides may be cytokines, such as interferons orinterleukins, or their receptors. Lack of expression of cytokines, or ofthe appropriate ones, has been implicated in susceptibility to diseases,and enhanced expression may lead to resistance to a number ofinfections. Expression patterns of cytokines may be altered to produce abeneficial effect, such as for example, a biasing of the immune responsetoward a Th1 type expression pattern, or a Th2 pattern in infection, orin autoimmune disease, wherein altered expression patterns may provebeneficial to the host.

In another embodiment, the therapeutic nucleic acid may encode or thetherapeutic polypeptide may be an enzyme, such as one involved inglycogen storage or breakdown. In another embodiment, the therapeuticprotein comprises a transporter, such as an ion transporter, for exampleCFTR, or a glucose transporter, or other transporters whose deficiency,or inappropriate expression, results in a variety of diseases.

In another embodiment, the therapeutic nucleic acid encodes or thetherapeutic polypeptide is a tumor suppressor or pro-apoptotic compound,which alters progression of cancer-related events.

In another embodiment, the therapeutic nucleic acid of the presentinvention may encode or the therapeutic polypeptide may be animmunomodulating protein. In one embodiment, the immunomodulatingprotein comprises cytokines, chemokines, complement or components, suchas interleukins 1 to 15, interferons alpha, beta or gamma, tumournecrosis factor, granulocyte-macrophage colony stimulating factor(GM-CSF), macrophage colony stimulating factor (M-CSF), granulocytecolony stimulating factor (G-CSF), chemokines such as neutrophilactivating protein (NAP), macrophage chemoattractant and activatingfactor (MCAF), RANTES, macrophage inflammatory peptides MIP-1a andMIP-1b, or complement components.

In another embodiment, a therapeutic nucleic acid of this invention mayencode or a therapeutic polypeptide may be a growth factor, ortissue-promoting factor. In one embodiment, the therapeutic compound isa bone morphogenetic protein, or OP-1, OP-2, BMP-5, BMP-6, BMP-2, BMP-3,BMP-4, BMP-9, DPP, Vg-1, 60A, or Vgr-1. In another embodiment, thetherapeutic nucleic acid encodes an RNA or peptide that facilitatesnerve regeneration or repair, and may include NGF, or other growthfactors. In another embodiment, the therapeutic polypeptide facilitatesnerve regeneration or repair, and may include NGF, or other growthfactors.

In another embodiment, the therapeutic nucleic acid may encode or thetherapeutic polypeptide may be natural or non-natural insulins,amylases, proteases, lipases, kinases, phosphatases, glycosyltransferases, trypsinogen, chymotrypsinogen, carboxypeptidases,hormones, ribonucleases, deoxyribonucleases, triacylglycerol lipase,phospholipase A2, elastases, amylases, blood clotting factors, UDPglucuronyl transferases, ornithine transcarbamoylases, cytochrome p450enzymes, adenosine deaminases, serum thymic factors, thymic humoralfactors, thymopoietins, growth hormones, somatomedins, costimulatoryfactors, antibodies, colony stimulating factors, erythropoietin,epidermal growth factors, hepatic erythropoietic factors(hepatopoietin), liver-cell growth factors, interleukins, interferons,negative growth factors, fibroblast growth factors, transforming growthfactors of the α family, transforming growth factors of the β family,gastrins, secretins, cholecystokinins, somatostatins, serotonins,substance P, transcription factors or combinations thereof.

In another embodiment, the gene comprises a reporter gene. In oneembodiment, the reporter gene encodes a fluorescent protein. In oneembodiment, the fluorescent protein is yECitrine or a yellow fluorescentprotein. In one embodiment, the fluorescent protein is the jellyfishgreen fluorescent protein, or a mutant or variant thereof. In anotherembodiment, the GMMOs specifically may comprise any gene other than areporter gene or a gene encoding a reporter protein.

In another embodiment, the reporter gene confers drug resistance. In oneembodiment, the reporter gene confers resistance to an antibiotic, suchas, for example, ampicilin, kanamycin, tetracycline, or others, as willbe appreciated by one skilled in the art. In another embodiment, theantibiotic resistance genes may include those conferring resistance toneomycin (neo), blasticidin, spectinomycin, erythromycin, phleomycin,Tn917, gentamycin, and bleomycin. An example of the neomycin resistancegene is the neomycin resistance gene of transposon Tn5 that encodes forneomycin phosphotransferase 11, which confers resistance to variousantibiotics, including G418 and kanamycin. In another embodiment, thereporter is a chloramphenicol acetyl transferase gene (cat) and confersresistance to chloramphenicol.

In one embodiment, the formulations and methods of this invention arefor prevention of, or therapeutic intervention of viral infection, or inanother embodiment, bacterial, parasitic, or fungal infection, or acombination thereof.

According to this aspect of the invention, the formulations and methodsof this invention are for prevention of, or therapeutic intervention indisease. In one embodiment, the disease for which the subject is thustreated may comprise, but is not limited to: muscular dystrophy, cancer,cardiovascular disease, hypertension, infection, renal disease,neurodegenerative disease, such as Alzheimer's disease, Parkinson'sdisease, Huntington's chorea, Creurtfeld-Jacob disease, autoimmunedisease, such as lupus, rheumatoid arthritis, endocarditis, Graves'disease or ALD, respiratory disease such as asthma or cystic fibrosis,bone disease, such as osteoporosis, joint disease, liver disease,disease of the skin, such as psoriasis or eczema, ophthalmic disease,otolaryngeal disease, other neurological disease such as Turretsyndrome, schizophrenia, depression, autism, or stoke, or metabolicdisease such as a glycogen storage disease or diabetes. It is to beunderstood that any disease whereby expression of a particular protein,provision of a therapeutic protein, provision of a drug, inhibition ofexpression of a particular protein, etc., which can be accomplished viathe formulations of this invention and according to the methods of thisinvention, is to be considered as part of this invention.

In one embodiment, the formulations and methods of the instant inventioncomprise a nucleic acid sequence operably linked to one or moreregulatory sequences. In one embodiment, a nucleic acid moleculeintroduced into a cell of a micro-organ is in a form suitable forexpression in the cell of the gene product encoded by the nucleic acid.Accordingly, in one embodiment, the nucleic acid molecule includescoding and regulatory sequences required for transcription of a gene (orportion thereof). When the gene product is a protein or peptide, thenucleic acid molecule includes coding and regulatory sequences requiredfor translation of the nucleic acid molecule include promoters,enhancers, polyadenylation signals, sequences necessary for transport ofan encoded protein or peptide, for example N-terminal signal sequencesfor transport of proteins or peptides to the surface of the cell orsecretion, in one embodiment.

Nucleotide sequences which regulate expression of a gene product (e.g.,promoter and enhancer sequences) are selected based upon the type ofcell in which the gene product is to be expressed and the desired levelof expression of the gene product. For example, a promoter known toconfer cell-type specific expression of a gene linked to the promotercan be used. A promoter specific for myoblast gene expression can belinked to a gene of interest to confer muscle-specific expression ofthat gene product. Muscle-specific regulatory elements which are knownin the art include upstream regions from the dystrophin gene (Klamut etal., (1989) Mol. Cell. Biol. 9:2396), the creatine kinase gene (Buskinand Hauschka, (1989) Mol. Cell. Biol. 9:2627) and the troponin gene (Marand Ordahl, (1988) Proc. Natl. Acad. Sci. USA. 85:6404). Negativeresponse elements in keratin genes mediate transcriptional repression(Jho Sh et al, (2001). J. Biol Chem). Regulatory elements specific forother cell types are known in the art (e.g., the albumin enhancer forliver-specific expression; insulin regulatory elements for pancreaticislet cell-specific expression; various neural cell-specific regulatoryelements, including neural dystrophin, neural enolase and A4 amyloidpromoters). Alternatively, a regulatory element which can directconstitutive expression of a gene in a variety of different cell types,such as a viral regulatory element, can be used. Examples of viralpromoters commonly used to drive gene expression include those derivedfrom polyoma virus, Adenovirus 2, cytomegalovirus (CMV) and Simian Virus40, and retroviral LTRs. Alternatively, a regulatory element whichprovides inducible expression of a gene linked thereto can be used. Theuse of an inducible regulatory element (e.g., an inducible promoter)allows for modulation of the production of the gene product in the cell.Examples of potentially useful inducible regulatory systems for use ineukaryotic cells include hormone-regulated elements (e.g., see Mader, S,and White, J. H. (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607),synthetic ligand-regulated elements (see, e.g., Spencer, D. M. et al1993) Science 262:1019-1024) and ionizing radiation-regulated elements(e.g., see Manome, Y. Et al. (1993) Biochemistry 32:10607-10613; Datta,R. et al. (1992) Proc. Natl. Acad. Sci. USA 89:1014-10153). Additionaltissue-specific or inducible regulatory systems which may be developedcan also be used in accordance with the invention.

In one embodiment, a regulatory sequence of the instant invention maycomprise a CMV promoter, while in another embodiment; the regulatorysequence may comprise a CAG promoter. In one embodiment, a CAG promoteris a composite promoter that combines the human cytomegalovirusimmediate-early enhancer and a modified chicken beta-actin promoter andfirst intron. In one embodiment, a regulatory sequence may comprise asimian virus (SV)-40 polyadenylation sequence, which in one embodiment,is the mechanism by which most messenger RNA molecules are terminated attheir 3′ ends in eukaryotes. In one embodiment, the polyadenosine(poly-A) tail protects the mRNA molecule from exonucleases and isimportant for transcription termination, for export of the mRNA from thenucleus, and for translation. In another embodiment, a formulation ofthe present invention may comprise one or more regulatory sequences. Inone embodiment, a regulatory sequence of this invention comprises SEQ IDNo. 12. In another embodiment, a regulatory sequence of this inventioncomprises SEQ ID No. 13.

In one embodiment, formulations of the instant invention comprising CMVor CAG promoters in conjunction with SV40 polyadenylation sequencedemonstrate long-term, high in vitro (FIGS. 1, 5, and 6B) and in vivo(FIG. 6A) expression levels of EPO and IFN-alpha. Without being bound bytheory, one factor that may contribute to the long-lasting, high levelsof gene product from micro-organs of the instant invention is the use ofCMV, or alternatively, CAG as a promoter, which may be especiallyeffective in micro-organ explants in promoting constitutive geneexpression.

In one embodiment, the term “promoter” refers to a DNA sequence, which,in one embodiment, is directly upstream of the coding sequence and isimportant for basal and/or regulated transcription of a gene. In oneembodiment, a promoter of the present invention is operatively linked toa gene of interest. In another embodiment, the promoter is a mutant ofthe endogenous promoter, which is normally associated with expression ofthe gene of interest, under the appropriate conditions.

In one embodiment, a promoter of the compositions and for use in themethods of the present invention is a regulatable promoter. In anotherembodiment, a regulatable promoter refers to a promoter wherebyexpression of a gene downstream occurs as a function of the occurrenceor provision of specific conditions which stimulate expression from theparticular promoter. In some embodiments, such conditions result indirectly turning on expression, or in other embodiments, removeimpediments to expression. In some embodiments, such conditions resultin turning off, or reducing expression.

In one embodiment, such conditions may comprise specific temperatures,nutrients, absence of nutrients, presence of metals, or other stimuli orenvironmental factors as will be known to one skilled in the art. In oneembodiment, a regulatable promoter may be regulated by galactose (e.g.UDP-galactose epimerase (GAL10), galactokinase (GAL1)), glucose (e.g.alcohol dehydrogenase II (ADH2)), or phosphate (e.g. acid phosphatase(PHO5)). In another embodiment, a regulatable promoter may be activatedby heat shock (heat shock promoter) or chemicals such as IPTG orTetracycline, or others, as will be known to one skilled in the art. Itis to be understood that any regulatable promoter and conditions forsuch regulation is encompassed by the vectors, nucleic acids and methodsof this invention, and represents an embodiment thereof.

In one embodiment, the formulations and methods of the instant inventionincrease the levels of a therapeutic polypeptide or nucleic acid by atleast 5% over basal levels. In another embodiment, the levels of atherapeutic polypeptide or nucleic acid are increased by at least 7%, inanother embodiment, by at least 10%, in another embodiment, by at least15%, in another embodiment, by at least 20%, in another embodiment, byat least 25%, in another embodiment, by at least 30%, in anotherembodiment, by at least 40%, in another embodiment, by at least 50%, inanother embodiment, by at least 60%, in another embodiment, by at least75%, in another embodiment, by at least 100%, in another embodiment, byat least 125%, in another embodiment, by at least 150% over basallevels, in another embodiment, by at least 200% over basal levels. Instill another embodiment, the formulations and methods of the instantinvention increase the level of a therapeutic polypeptide or nucleicacid upon administration, wherein the level of the therapeuticpolypeptide or nucleic acid then returns to basal or near basal levels.In one embodiment, the return to basal or near basal levels occurswithin one month of administration of the therapeutic peptide or nucleicacid.

In one embodiment, expression of a therapeutic polypeptide or nucleicacid via the formulation of the present invention is increased comparedto “basal levels”, which in one embodiment, are levels of the geneexpressed in hosts or cell culture that had not been administered orotherwise contacted with the therapeutic formulation of the presentinvention.

In another embodiment, the formulations and methods of the instantinvention increase the levels of a therapeutic polypeptide or nucleicacid to approximately 2000 ng/day, or in another embodiment, 1500ng/day, or in another embodiment, 1000 ng/day, or in another embodiment,750 ng/day, or in another embodiment, 500 ng/day, or in anotherembodiment, 250 ng/day, or in another embodiment, 150 ng/day, or inanother embodiment, 100 ng/day, or in another embodiment, 75 ng/day, orin another embodiment, 50 ng/day, or in another embodiment, 25 ng/day.In another embodiment, he formulations and methods of the instantinvention increase the levels of a therapeutic polypeptide to between20-70 mU/mL, or in another embodiment, 50-100 mU/mL, or in anotherembodiment, 5-20 mU/mL, or in another embodiment, 100-200 mU/mL, or inanother embodiment, 10-70 mU/mL, or in another embodiment, 5-80 mU/mL.In another embodiment, the formulations and methods of the instantinvention increase the levels of a therapeutic polypeptide to between500-1000 mU/mL, or in another embodiment, 250-750 mU/mL, or in anotherembodiment, 500-5000 mU/mL.

In one embodiment, the formulations and methods of the instant inventionincrease the levels of a functional marker, which in one embodiment, ishematocrit levels, by at least 5% over basal levels. In anotherembodiment, the levels of the functional marker are increased by atleast 7%, in another embodiment, by at least 10%, in another embodiment,by at least 15%, in another embodiment, by at least 20%, in anotherembodiment, by at least 25%, in another embodiment, by at least 30%, inanother embodiment, by at least 40%, in another embodiment, by at least50%, in another embodiment, by at least 60%, in another embodiment, byat least 75%, in another embodiment, by at least 100%, in anotherembodiment, by at least 125%, in another embodiment, by at least 150%over basal levels, in another embodiment, by at least 200% over basallevels.

In one embodiment, the therapeutic formulation of the present inventionis “long-lasting”, which in one embodiment refers to a formulation thatcan increase secretion, expression, production, circulation orpersistence of a therapeutic polypeptide or nucleic acid In oneembodiment, expression levels of a therapeutic polypeptide or nucleicacid are increased over basal levels for at least one month, or inanother embodiment, for at least six months. In one embodiment, theincrease is for 90% of the month. In another embodiment, the increase isfor 90% of six months. In yet another embodiment, the increase is for90% of any time period measured. In still another embodiment, theincrease is for 80% of the time period measured. In a furtherembodiment, the increase is for 70% of the time period measured. Inanother embodiment, the increase is for 60% of the time period measured.In a yet another embodiment, the increase is for 50% of the time periodmeasured.

In another embodiment, the therapeutic formulation of the presentinvention is “long-lasting”, which in one embodiment refers to aformulation that can increase secretion, expression, production,circulation or persistence of a target molecule of the therapeuticpolypeptide or nucleic acid. For instance, a target molecule may be apolypeptide, an RNA, a glycoprotein, a peptide, a glycosaminoglycan,protein-RNA complex, a DNA, or any downstream molecule synthesized by acell, or any combination thereof. For example, when the therapeuticpolypeptide is erythropoietin, a target molecule may be Hb. Accordingly,in one embodiment, a long-lasting formulation of the present inventionmay increase production, circulation or persistence of Hb in a subject.

In one embodiment, increase of a target molecule over basal levels isfor at least one month, or in another embodiment, for at least sixmonths. In one embodiment, the increase is for 90% of the month. Inanother embodiment, the increase is for 90% of six months. In yetanother embodiment, the increase is for 90% of any time period measured.In still another embodiment, the increase is for 80% of the time periodmeasured, e.g., one month, six months or a year. In a furtherembodiment, the increase is for 70% of the time period measured, e.g.,one month, six months or a year. In another embodiment, the increase isfor 60% of the time period measured, e.g., one month, six month or ayear. In a yet another embodiment, the increase is for 50% of the timeperiod measured, e.g., one month, six months or a year.

In yet another embodiment of the invention, the therapeutic formulationof the present invention is “long-lasting”, which refers to aformulation that can increase secretion, expression, production,circulation or persistence of a functional marker. In one embodiment,the functional marker is hematocrit. In another embodiment, thefunctional marker is Hb. In yet another embodiment, the levels of afunctional marker, for example hematocrit or Hb, are increased for atleast 2 weeks, in another embodiment, for at least 3 weeks, in anotherembodiment, for at least 4 weeks, in another embodiment, for at least 5weeks, in another embodiment, for at least 6 weeks, in anotherembodiment, for at least 8 weeks, in another embodiment, for at least 2months, in another embodiment, for at least 2 months in anotherembodiment, for at least 2 months in another embodiment, for at least 3months in another embodiment, for at least 4 months, in anotherembodiment, for at least 5 months, in another embodiment, for at least 7months, in another embodiment, for at least 8 months, in anotherembodiment, for at least 9 months, in another embodiment, for at least10 months, in another embodiment, for at least 11 months, or, in anotherembodiment, for at least 1 year. In another embodiment, expressionlevels of a therapeutic polypeptide or nucleic acid are increased for atleast 4-6 months.

In one embodiment, increase of the functional marker, e.g., Hb, overbasal levels is reflected in 90% of measurements made during any timeperiod. In still another embodiment, the increase is reflected in 80% ofthe measurements made during any time period, e.g., one month, sixmonths or a year. In a further embodiment, the increase is reflected in70% of the measurements made during any time period, e.g., one month,six months or a year. In another embodiment, the increase is reflectedin 60% of the measurements made during any time period, e.g., one month,six month or a year. In a yet another embodiment, the increase isreflected in 50% of the measurements made during any time period, e.g.,one month, six months or a year.

In one embodiment, the nucleic acid sequence encoding a therapeuticpolypeptide or nucleic acid is optimized for increased levels oftherapeutic polypeptide or nucleic acid expression, or, in anotherembodiment, for increased duration of therapeutic polypeptide or nucleicacid expression, or, in another embodiment, a combination thereof.

In one embodiment, the term “optimized” refers to a desired change,which, in one embodiment, is a change in gene expression and, in anotherembodiment, in protein expression. In one embodiment, optimized geneexpression is optimized regulation of gene expression. In anotherembodiment, optimized gene expression is an increase in gene expression.According to this aspect and in one embodiment, a 2-fold through1000-fold increase in gene expression compared to wild-type iscontemplated. In another embodiment, a 2-fold to 500-fold increase ingene expression, in another embodiment, a 2-fold to 100-fold increase ingene expression, in another embodiment, a 2-fold to 50-fold increase ingene expression, in another embodiment, a 2-fold to 20-fold increase ingene expression, in another embodiment, a 2-fold to 10-fold increase ingene expression, in another embodiment, a 3-fold to 5-fold increase ingene expression is contemplated.

In another embodiment, optimized gene expression may be an increase ingene expression under particular environmental conditions. In anotherembodiment, optimized gene expression may comprise a decrease in geneexpression, which, in one embodiment, may be only under particularenvironmental conditions.

In another embodiment, optimized gene expression is an increasedduration of gene expression. According to this aspect and in oneembodiment, a 2-fold through 1000-fold increase in the duration of geneexpression compared to wild-type is contemplated. In another embodiment,a 2-fold to 500-fold increase in the duration of gene expression, inanother embodiment, a 2-fold to 100-fold increase in the duration ofgene expression, in another embodiment, a 2-fold to 50-fold increase inthe duration of gene expression, in another embodiment, a 2-fold to20-fold increase in the duration of gene expression, in anotherembodiment, a 2-fold to 10-fold increase in the duration of geneexpression, in another embodiment, a 3-fold to 5-fold increase in theduration of gene expression is contemplated. In another embodiment, theincreased duration of gene expression is compared to gene expression innon-vector-expressing controls, or alternatively, compared to geneexpression in wild-type-vector-expressing controls.

Expression in mammalian cells is hampered, in one embodiment, bytranscriptional silencing, low mRNA half-life, alternative splicingevents, premature polyadenylation, inefficient nuclear translocation andavailability of rare tRNAs pools. The source of many problems inmammalian expressions is found within the message encoding the transgeneincluding in the autologous expression of many crucial mammalian genesas well. The optimization of mammalian RNAs may include modification ofcis acting elements, adaptation of its GC-content, modifying codon biaswith respect to non-limiting tRNAs pools of the mammalian cell, avoidinginternal homologous regions and excluding RNAi's.

Therefore, in one embodiment, when relying on carefully designedsynthetic genes, stable messages with prolonged half-lives, constitutivenuclear export and high level protein production within the mammalianhost can be expected.

Thus, in one embodiment, optimizing a gene entails adapting the codonusage to the codon bias of host genes, which in one embodiment, are Homosapiens genes; adjusting regions of very high (>80%) or very low (<30%)GC content; avoiding one or more of the following cis-acting sequencemotifs: internal TATA-boxes, chi-sites and ribosomal entry sites;AT-rich or GC-rich sequence stretches; ARE, INS, CRS sequence elements;repeat sequences and RNA secondary structures; (cryptic) splice donorand acceptor sites, branch points; or a combination thereof. In oneembodiment, a gene is optimized for expression in homo sapien cells. Inanother embodiment, a gene is optimized for expression in micro-organs.In yet another embodiment, a gene is optimized for expression in dermalcells. In still another embodiment, optimizing a gene expression entailsadding sequence elements to flanking regions of a gene and/or elsewherein the expression vector. Sequence elements that may be added foroptimizing gene expression include, for example, matrix-attached regions(MAR), specialized chromatin structures (SCS) and woodchuck hepatitispost-transcriptional regulatory elements (WPRE).

In one embodiment, as demonstrated herein, optimized genes, such as EPO,maintain an increase percent of peak expression levels for an extendedperiod of time compared to both non-optimized EPO expressed from agutless adenovirus vector or non-optimized EPO expressed from anadenovirus 5 vector (FIGS. 3 and 4).

In one embodiment, the term “gene” refers to a nucleic acid fragmentthat is capable of being expressed as a specific protein, includingregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure.

In one embodiment, the therapeutic nucleic acid may be any gene whichencodes an RNA molecule (sense or antisense), peptide, polypeptide,glycoprotein, lipoprotein or combination thereof or to any other postmodified polypeptide. In one embodiment of the invention, the gene ofinterest may be naturally expressed in the tissue sample. In anotherembodiment of this invention, the tissue sample may be geneticallyengineered so that at least one cell will express the gene of interest,which is either not naturally expressed by the cell or has an alteredexpression profile within the cell. In one embodiment, the therapeuticnucleic acid of the present invention may encode or the therapeuticpolypeptide may be any of the proteins listed in U.S. patent applicationSer. No. 10/376,506, which is incorporated herein by reference in itsentirety.

In one embodiment, the genetically modified micro-organ is a geneticallymodified dermal micro-organ. “Dermal” micro-organs (“DMO”) may comprisea plurality of dermis components, where in one embodiment; dermis is theportion of the skin located below the epidermis. These components maycomprise skin fibroblast, epithelial cells, other cell types, bases ofhair follicles, nerve endings, sweat and sebaceous glands, and blood andlymph vessels. In one embodiment, a dermal micro-organ may comprise fattissue, wherein in another embodiment, a dermal micro-organ may notcomprise fat tissue.

In some embodiments of the invention, the dermal micro-organ may containtissue of a basal epidermal layer and, optionally, other epidermallayers of the skin. In other embodiments, the dermal micro-organ doesnot include basal layer tissue. In another embodiment of the invention,the dermal micro-organ does not include epidermal layers. In yet anotherembodiment, the dermal micro-organ contains an incomplete epidermallayer. In still another embodiment, the dermal micro-organ may contain afew layers of epidermal tissue. In still another embodiment, the dermalmicro-organ may contain invaginations of the epidermis into the dermis.In a further embodiment, the dermal micro-organ may include additionalcomponents such as sweat glands and/or hair follicles.

In one embodiment of the invention, the DMO includes the entirecross-section of the dermis. In another embodiment of the invention, thedermal micro-organ includes part of the cross-section of the dermis. Ina further embodiment, the DMO includes most of the cross section of thedermis, namely, most of the layers and components of the dermisincluding the papillary and reticular dermis. In a further embodiment,the DMO includes primarily dermal tissue, but may also include fattissue. In some embodiments of the invention, the DMO does not producekeratin or produces a negligible amount of keratin, thereby preventingthe formation of keratin cysts following implantation in a recipient,for example, following subcutaneous or intradermal implantation. Furtherdetails regarding dermal micro-organs, including methods of harvesting,maintaining in culture, and implanting said dermal micro-organs, aredescribed in PCT Patent Application WO2004/099363, which is incorporatedherein by reference in its entirety.

In another embodiment, the invention provides a method of providing atherapeutic polypeptide to a subject in need over a sustained periodcomprising providing one or more genetically modified micro-organs, saidmicro-organs comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences; and implanting saidgenetically modified micro-organ in said subject, wherein said nucleicacid sequence encodes a therapeutic polypeptide and whereby theexpression level of the therapeutic nucleic acid or polypeptide isincreased by more than 5% over basal level and said increase ismaintained for greater than one month. In another embodiment, theinvention provides a method of providing a therapeutic polypeptide to asubject in need over a sustained period comprising providing one or moregenetically modified micro-organs, said micro-organs comprising a vectorcomprising a nucleic acid sequence operably linked to one or moreregulatory sequences; and implanting said genetically modifiedmicro-organ in said subject, wherein said nucleic acid sequence encodesa therapeutic polypeptide and wherein said vector is a helper-dependentadenovirus vector. In another embodiment, the invention provides amethod of providing a therapeutic polypeptide to a subject in need overa sustained period comprising providing one or more genetically modifiedmicro-organs, said micro-organs comprising a vector comprising a nucleicacid sequence operably linked to one or more regulatory sequences; andimplanting said genetically modified micro-organ in said subject,wherein said nucleic acid sequence encodes a therapeutic polypeptide andwherein said vector is a helper-dependent adenovirus vector.

In another embodiment, the methods described hereinabove provide atherapeutic nucleic acid to a subject in need wherein the expressionlevel of the therapeutic nucleic acid or polypeptide is increased bymore than 5% over basal level and said increase is maintained forgreater than one hour, 3 hours, 6 hours, 9 hours, 12 hours, 18 hours, 1day, or 2 days, wherein said vector is a helper-dependent adenovirusvector, or a combination thereof.

In one embodiment, this invention provides a therapeutic formulation asdescribed hereinabove in which the therapeutic polypeptide iserythropoietin or wherein the therapeutic nucleic acid encodeserythropoietin. In another embodiment, this invention provides along-lasting erythropoietin formulation comprising a geneticallymodified micro-organ, said micro-organ comprising a vector comprising anucleic acid sequence operably linked to one or more regulatorysequences, wherein said nucleic acid sequence encodes erythropoietin andwhereby said formulation increases erythropoietin levels by more than 5%over basal levels and said increased erythropoietin levels persist forgreater than one month. In another embodiment, the formulation increaseserythropoietin levels by more than 5% over basal levels and the increaseerythropoietin levels persist for less than one month. In yet anotherembodiment, the invention provides a method of providing a therapeuticformulation to a subject in need in which the therapeutic polypeptide iserythropoietin or wherein the therapeutic nucleic acid encodeserythropoietin. In still another embodiment, the invention provides amethod of providing erythropoietin to a subject in need.

In a further embodiment, this invention provides a method of deliveringerythropoietin to a subject in need over a sustained period comprising:providing one or more genetically modified micro-organs, saidmicro-organs comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences; and implanting saidgenetically modified micro-organ in said subject, wherein said nucleicacid sequence encodes erythropoietin and whereby erythropoietin levelsare increased by more than 5% over basal levels and said increasederythropoietin levels persist for greater than one month. In anotherembodiment, the erythropoietin levels are increased by more than 5% overbasal levels and the increased erythropoietin levels persist for lessthan one month.

In yet another embodiment, this invention provides a method of inducingformation of new blood cells in a subject in need over a sustainedperiod comprising: providing one or more genetically modifiedmicro-organs, said micro-organs comprising a vector comprising a nucleicacid sequence operably linked to one or more regulatory sequences; andimplanting said genetically modified micro-organ in said subject,wherein said nucleic acid sequence encodes erythropoietin and wherebyerythropoietin levels are increased by more than 5% over basal levelsand said increased erythropoietin levels persist for greater than onemonth. In still another embodiment, the erythropoietin levels areincreased by more than 5% over basal levels and the increasederythropoietin levels persist for less than one month.

Erythropoietin (EPO) is a glycoprotein hormone involved in thematuration of erythroid progenitor cells into erythrocytes. In oneembodiment, erythropoietin is essential in regulating levels of redblood cells in circulation. Naturally occurring erythropoietin isproduced by the kidneys and liver, circulates in the blood, andstimulates the production of red blood cells in bone marrow, in oneembodiment, in response to hypoxia.

In one embodiment, EPO of the compositions and methods of the instantinvention may comprise glycosylation patterns similar to those of EPOextracted from human or animal urine, or in another embodiment, plasma.

The identification, cloning, and expression of genes encodingerythropoietin are described in U.S. Pat. Nos. 5,756,349; 5,955,422;5,618,698; 5,547,933; 5,621,080; 5,441,868; and 4,703,008, which areincorporated herein by reference. A description of the purification ofrecombinant erythropoietin from cell medium that supported the growth ofmammalian cells containing recombinant erythropoietin plasmids forexample, are included in U.S. Pat. No. 4,667,016 to Lai et al, which isincorporated herein by reference. Recombinant erythropoietin produced bygenetic engineering techniques involving the expression of a proteinproduct in vitro from a host cell transformed with the gene encodingerythropoietin has been used to treat anemia resulting from chronicrenal failure.

EPO may be used in the treatment of anemia as a result of renal failureincluding chronic kidney disease (CKD) and end stage renal disease(ESRD); anemia associated with HIV infection in zidovudine (AZT) treatedpatients; anemia associated with cancer chemotherapy; microangiopathichaemolytic anemia that may be a secondary to mechanical valvehaemolysis; anemia of prematurity; anemia as a result rheumatoidarthritis and other inflammatory conditions; and anemia associated withcancer including multiple myeloma and non-Hodgkin lymphoma. In addition,administration of EPO may benefit subjects prior to scheduled surgery,subjects suffering from hematopoietic stem cell disorders, subjects inneed of acceleration of erythroid repopulation after bone marrowtransplantation, or subjects in need of induction of fetal Hb synthesisas a result of sickle cell anemia and thalassemia.

Administration of rhu-EPO (recombinant human-EPO) has become routine inthe treatment of anemia secondary to renal insufficiency, where doses of50-150 u/kg given three times per week are used to gradually restorehematocrit and eliminate transfusion dependency. This results in anaverage daily dosage of 21.4-64.3 EPO U/Kg/day to a subject beingtreated.

Many cell surface and secretory proteins produced by eukaryotic cellsare modified with one or more oligosaccharide groups calledglycosylation, which can dramatically affect protein stability,secretion, and subcellular localization as well as biological activity.In one embodiment, both human urinary derived erythropoietin andrecombinant erythropoietin (expressed in mammalian cells) having theamino acid sequence 1-165 of human erythropoietin comprise threeN-linked and one O-linked oligosaccharide chains which together compriseabout 40% of the total molecular weight of the glycoprotein. In oneembodiment, non-glycosylated erythropoietin has greatly reduced in vivoactivity compared to the glycosylated form but does retain some in vitroactivity. In one embodiment, the EPO of the compositions and for use inthe methods of the present invention are fully glycosylated, while inanother embodiment, they are comprise some glycosylated residues, whilein another embodiment, they are not glycosylated.

In one embodiment, the EPO gene may be a wild-type EPO gene, while inanother embodiment, the EPO gene may be modified. In one embodiment, themodified EPO gene may be optimized.

In one embodiment, the EPO gene has a nucleic acid sequence thatcorresponds to that set forth in Genbank Accession Nos: X02158;AF202312; AF202311; AF202309; AF202310; AF053356; AF202306; AF202307; orAF202308 or encodes a protein sequence that corresponds to that setforth in Genbank Accession Nos: CAA26095; AAF23134; AAF17572; AAF23133;AAC78791; or AAF23132. In another embodiment, the EPO precursor gene hasa nucleic acid sequence that corresponds to that set forth in GenbankAccession Nos: NM_(—)000799; M11319; BC093628; or BC111937 or encodes aprotein sequence that corresponds to that set forth in Genbank AccessionNos: NP_(—)000790; AAA52400; AAH93628; or AAI11938. In anotherembodiment, the EPO gene has a nucleic acid sequence as presented in SEQID No: 7, while in another embodiment, the EPO gene has an amino acidsequence as presented in SEQ ID No: 10. In another embodiment, the EPOgene has a nucleic acid that is homologous to that presented in SEQ IDNo: 7, while in another embodiment, the EPO gene has an amino acidsequence that is homologous to that presented in SEQ ID No: 10. In oneembodiment, the nucleic acid encoding erythropoietin operably linked toone or more regulatory sequences has a nucleic acid sequence aspresented in SEQ ID No: 11. In another embodiment, the nucleic acidencoding erythropoietin operably linked to one or more regulatorysequences has a nucleic acid sequence that is homologous to thatpresented in SEQ ID No: 11. In one embodiment, the vector used forgenetic modification has a nucleic acid sequence as presented in SEQ IDNo. 3. In another embodiment, the vector used for genetic modificationhas a nucleic acid sequence as presented in SEQ ID No. 4. In yet anotherembodiment, the vector used for genetic modification has a nucleic acidsequence homologous to that presented in SEQ ID No. 3. In still anotherembodiment, the vector used for genetic modification has a nucleic acidsequence homologous to that presented in SEQ ID No. 4.

In one embodiment, the formulations of the present invention may be usedto treat a subject having anemia. In one embodiment, anemia is definedas “a pathologic deficiency in the amount of oxygen-carrying Hb in thered blood cells.” Symptoms of anemia include fatigue, diminished abilityto perform daily functions, impaired cognitive function, headache,dizziness, chest pain and shortness of breath, nausea, depression, pain,or a combination thereof. In one embodiment, anemia is associated with apoorer prognosis and increased mortality.

In one embodiment, administration of a GMMO-hEPO of this inventionincreases and/or maintains Hb to therapeutic levels. In someembodiments, administration of a GMMO-hEPO results in increased and/ormaintained Hb for extended time periods. In certain instances, themeasurable EPO levels during the time period of increased and/ormaintained Hb levels do not show a comparable percent increase. In oneembodiment, administration of a GMMO-hEPO of this invention increasesand/or maintains hematocrit levels. In some embodiments, administrationof a GMMO-hEPO results in increased and/or maintained hematocrit levelsfor extended time periods.

Anemia is often a consequence of renal failure due to decreasedproduction of erythropoietin from the kidney. In another embodiment,anemia is caused by lowered red blood cell (erythroid) production bybone marrow due to cancer infiltration, lymphoma or leukemia includingnon-Hodgkin's lymphoma, multiple myeloma, chemotherapy, mechanical valvehaemolysis, prematurity, rheumatoid arthritis, inflammatory conditions,hematopoietic disorders, sickle cell anemia, thalassemia or marrowreplacement. Other causes of anemia comprise, blood loss due toexcessive bleeding such as hemorrhages or abnormal menstrual bleeding;cancer therapies such as surgery, radiotherapy, chemotherapy e.g.,treatment of AIDs patients with Zidovudine (AZT), immunotherapy, or acombination thereof; infiltration or replacement of cancerous bonemarrow; increased hemolysis, which in one embodiment is breakdown ordestruction of red blood cells; low levels of erythropoietin, or acombination thereof. In one embodiment, anemia refers to Fanconi anemia,which in one embodiment is an inherited anemia that leads to bone marrowfailure (aplastic anemia) and often to acute myelogenous leukemia (AML).In another embodiment, anemia refers to Diamond Blackfan anemia,normocytic anemia, aplastic anemia, iron-deficiency anemia, vitamindeficiency anemia, Sideroblastic Anemia, Paroxysmal NocturnalHemoglobinuria, Anemia of Chronic Disease, Anemia in Kidney Disease andDialysis, or a combination thereof.

In another embodiment, the long-lasting EPO formulation of the instantinvention is used for treating a diabetic subject. According to thisaspect and in one embodiment, the EPO formulation of the instantinvention may be used in conjunction with other treatments for diabetesknown in the Art, including, inter alia, insulin administration, oralhypoglycemic drugs, which in one embodiment are sulfonurea drugs, whichin one embodiment including inter alia glucotrol, glyburide, glynase andamaryl; glucophage, thiazolidinediones including inter alia rezulin,actos and avandia; or a combination thereof. In another embodiment, thelong-lasting EPO formulation of the instant invention is used fortreating a subject suffering from chronic kidney disease, while inanother embodiment, is used for treating a subject suffering fromend-stage renal disease. In another embodiment, the formulations of theinstant invention are used for subjects that are susceptible to theabove-mentioned diseases or conditions.

In yet another embodiment, the long-lasting EPO formulation of theinstant invention is used for increasing hemoglobin level in a subject.A subject in need of increased Hb, may for instance be a subject priorto major surgery.

It is to be understood that the formulations and methods of thisinvention may be used to treat anemia, regardless of the cause of anemiaand whether or not the cause of anemia is known.

In one embodiment, the formulations and methods thereof provide aneffective EPO therapy.

By the term “effective EPO therapy” it is meant a level of EPOsufficient to bring the Hb level in a patient within the therapeuticwindow. In one embodiment, “effective EPO therapy” refers to an increasein erythropoiesis in a subject in need. In one embodiment, “effectiveEPO therapy” refers to prevention of a decrease of erythropoiesis in asubject in need. As used herein, the term “effective EPO therapy” mayalso be referred to herein as an “effective dosage” or “effective dose”of erythropoietin.

In one embodiment of the invention, effective EPO therapy is achieved byimplanting at least one (1) human EPO-GMMO (hEPO-GMMO) into a patient.In another embodiment, effective EPO therapy is achieved by implantingat least two (2) hEPO-GMMO into a patient. In one embodiment, effectiveEPO therapy is achieved by implanting at least three (3) hEPO-GMMO intoa patient. In one embodiment, effective EPO therapy is achieved byimplanting at least four (4) hEPO-GMMO into a patient. In oneembodiment, effective EPO therapy is achieved by implanting at leastfive (5) hEPO-GMMO into a patient. In one embodiment, effective EPOtherapy is achieved by implanting at least six (6) hEPO-GMMO into apatient. In one embodiment, effective EPO therapy is achieved byimplanting greater than six (6) hEPO-GMMO into a patient.

As used herein, the term “therapeutic window” it is meant the desiredlevel of Hb in a subject in need. In one embodiment, the therapeuticwindow refers to a Hb concentration within the range of 10 gm/dl to 12gm/dl. In another embodiment, the therapeutic window refers to a Hbconcentration within the range of 9-11 gm/dl. In yet another embodiment,the Hb concentration is within the range of 9.5-12.6 gm/dl. In stillanother embodiment, the Hb concentration is within the range of 9-13.2gm/dl. In a further embodiment, the Hb concentration is within the rangeof 8.5-13.8 gm/dl. In another embodiment, the Hb concentration is withinthe range of 8-14.4 gm/dl. As used herein, the term “therapeutic window”may also be referred to as “physiological levels” or “physiologicalhemoglobin levels”.

In one embodiment, the subject in need is human. In one embodiment, thesubject in need is a renal patient. In another embodiment, the subjectin need is suffering from end-stage renal disease. In yet anotherembodiment, the subject in need is suffering from chronic kidney disease(CKD). In still another embodiment, the subject is suffering frompre-dialysis CKD. In another embodiment, the subject in need isdiabetic. In one embodiment, the subject is a pre-dialysis patient. Inanother embodiment, the subject is concurrently receiving dialysis. Inyet another embodiment, the subject has received dialysis.

In one embodiment of the invention, an effective EPO therapy brings theHb (“Hb”) level in a patient within the therapeutic window. In anotherembodiment of the invention, an effective EPO therapy brings the Hblevel in a patient within a range +/−10% of the therapeutic window. Inanother embodiment of the invention, an effective EPO therapy brings theHb level in a patient within a range +/−20% of the therapeutic window.In yet another embodiment of the invention, an effective EPO therapybrings the Hb level in a patient within a range +/−30% of thetherapeutic window. In still another embodiment, an effective EPOtherapy brings the Hb level in a patient within a range +/−40% of thetherapeutic window. In a further embodiment, an effective EPO therapybrings the Hb level in a patient within a range +/−50% of thetherapeutic window.

In one embodiment, an increase of blood Hb levels above 11.5 g/dl forfour consecutive weekly measurements, may be considered outside of thetherapeutic window. In another embodiment, an increase of blood Hblevels above 12.0 g/dl for four consecutive weekly measurements, may beconsidered outside of the therapeutic window. In an effort to avoid anincrease of blood Hb levels outside of the therapeutic window, methodsof implantation of a long-lasting erythropoietin formulation may bedirected to avoid a resultant elevation of serum EPO above the upperlimit of the normal physiological range, defined as a level exceeding200 mU/ml.

As used herein, the terms “effective dosage” or “effective dose” refersto the effective amount of a therapeutic polypeptide expressed from anat least one GMMO per day.

As used herein, units for EPO are the accepted International units andare referred to herein using the symbol “U” or “IU”.

In one embodiment, dosage for an effective EPO therapy is between 18-150IU/kg bodyweight of a patient/day. In another embodiment, effective EPOtherapy is between 12-150 U/kg bodyweight of a patient/day. In oneembodiment, dosage for an effective EPO therapy is between 20-40 IU/kgbodyweight of a patient/day. In one embodiment, dosage for an effectiveEPO therapy is between 40-60 IU/kg bodyweight of a patient/day. In oneembodiment, dosage for an effective EPO therapy is between 60-80 IU/kgbodyweight of a patient/day. In one embodiment, dosage for an effectiveEPO therapy is between 80-100 IU/kg bodyweight of a patient/day. In oneembodiment, dosage for an effective EPO therapy is between 100-120 IU/kgbodyweight of a patient/day. In one embodiment, dosage for an effectiveEPO therapy is between 120-150 IU/kg bodyweight of a patient/day.

In another embodiment, dosage for an effective EPO therapy is between18-25 IU/kg bodyweight/day (low dose). In yet another embodiment, dosagefor an effective EPO therapy is between 18-30 IU/kg bodyweight/day (lowdose). In one embodiment, dosage for an effective EPO therapy is between35-45 IU/kg bodyweight/day (mid dose). In still another embodiment,dosage for an effective EPO therapy is between 30-50 IU/kgbodyweight/day (mid dose). In one embodiment, dosage for an effectiveEPO therapy is between 55-65 IU/kg bodyweight/day (high dose). In afurther embodiment, dosage for an effective EPO therapy is between 50-65IU/kg bodyweight/day (high dose).

In one embodiment, dosage for an effective EPO therapy is 20 IU/kgbodyweight of a patient/day. In one embodiment, dosage for an effectiveEPO therapy is 40 IU/kg bodyweight of a patient/day. In one embodiment,dosage for an effective EPO therapy is 60 IU/kg bodyweight of apatient/day. In one embodiment, dosage for an effective EPO therapy is80 IU/kg bodyweight of a patient/day. In one embodiment, dosage for aneffective EPO therapy is 100 IU/kg bodyweight of a patient/day. In oneembodiment, dosage for an effective EPO therapy is 120 IU/kg bodyweightof a patient/day. In one embodiment, dosage for an effective EPO therapyis 150 IU/kg bodyweight of a patient/day. In one embodiment, dosage isnot to exceed about 65 IU/kg bodyweight/day.

In alternative embodiments, effective doses may be tailored to eachsubject individually, taking into account the patient's weight,historical Hb levels and average EPO dose previously administered by ESAinjections. The average EPO dose previously administered may becalculated from the time period of one month prior to a method ofimplantation. Alternatively, the time period for calculation of anaverage EPO dosage previously administered may be calculated from a timeperiod greater or less than at least on month. Dosage may be based onthe amount of EPO administered during the at least one month prior toimplantation, wherein the dosage administered is normalized to a dailybasis. In some circumstances, the dosage may be based on the amount ofEPO administered during at least a two month time period prior toimplantation. In certain circumstances, the dosage may be based on theamount of EPO administered during at least a three month time periodprior to implantation. In other circumstances, the dosage may be basedon the amount of EPO administered during at least a six month timeperiod prior to implantation. For example, if a subject previouslyreceived three injections per week totaling 150 U/kg/week, a tailoreddosage may include implantation of at least one genetically modifiedmicro-organ producing approximately 20 U/Kg/day.

In one embodiment, the dosage matches the amount a subject previouslyreceived, normalized to a daily bases. In another embodiment, the dosageis reduced by up to 25% of the amount a subject previously received,normalized to a daily bases. In yet another embodiment, the dosage isincreased by up to 25% of the amount a subject previously received,normalized to a daily bases. In a further embodiment, the dosage isreduced by up to 50% of the amount a subject previously received,normalized to a daily bases. In yet another embodiment, the dosage isincreased by up to 50% of the amount a subject previously received,normalized to a daily bases.

In one embodiment, response to implantation of an at least one hEPO-GMMOis sustained elevation of Hb levels for at least one month. In oneembodiment, response to implantation of an at least one hEPO-GMMO issustained elevation of Hb levels for at least two months. In oneembodiment, response to implantation of an at least one hEPO-GMMO issustained elevation of Hb levels for at least three months. In oneembodiment, response to implantation of an at least one hEPO-GMMO issustained elevation of Hb levels for at least four months. In oneembodiment, response to implantation of an at least one hEPO-GMMO issustained elevation of Hb levels for at least five months. In oneembodiment, response to implantation of an at least one hEPO-GMMO issustained elevation of Hb levels for at least six months. In oneembodiment, response to implantation of an at least one hEPO-GMMO issustained elevation of Hb levels for greater than six months. In oneembodiment, response to implantation of an at least one hEPO-GMMO issustained elevation of Hb levels for greater than nine months. In oneembodiment, response to implantation of an at least one hEPO-GMMO issustained elevation of Hb levels for greater than one year.

It may be that the response to implantation of an at least one hEPO-GMMOdoes not sustain elevation of Hb or provide sufficient elevation. Insuch a case, additional hEPO-GMMO may be implanted in the subject. Forexample, if following implantation of at least one hEPO-GMMO the bloodHb level decreases by about 1 g/dl or more per day, or per week, or pertwo weeks or per month, compared to the baseline Hb average during a onemonth period prior to implantation, additional hEPO-GMMO may beimplanted. Alternatively, if following implantation of at least onehEPO-GMMO the blood Hb level decreases by about 1 g/dl or more per day,or per week, or per two weeks or per month, compared to an initialincreased average Hb following implantation, additional hEPO-GMMO may beimplanted. In one embodiment, additional hEPO-GMMO may provide up to 25%increased EPO IU. In another embodiment, additional hEPO-GMMO mayprovide up to 50% increased EPO IU.

Treatment implanting long-lasting erythropoietin formulations, e.g.,genetically modified micro-organs expressing and secreting EPO, aims tosupply a steady continuous production and delivery of EPO to patients inneed. Patients in need may include those suffering from anemia and/orthose in need of increased Hb. In certain instances, anemic subjects orthose in need of increased Hb may benefit from treatment with a morephysiological EPO treatment [Fishbane, S., Recombinant HumanErythropoietin: Has Treatment Reached its Full Potential, Seminars inDialysis, Vol 19, No 1, 2006, pp. 1-4] Implantation of autologousgenetically modified micro-organs secreting and expressing EPO back intoa patient, wherein the autologous tissue remains localized and suppliessustained treatment, may provide this benefit. A strong advantage ofthis method is that if the delivered dose of EPO is too high, or if thetreatment needs to be terminated for any reason, one or more of theimplanted genetically modified micro-organs may be simply removed (oreven potentially ablated in situ) in order to stop the production anddelivery of the EPO. In one embodiment, if the blood level of Hb isgreater than 11.5 g/dl, then at least one hEPO-GMMO may be removed orinactivated to reduce the EPO U by about 25%. In another embodiment, ifthe blood level of Hb is greater than 12.0 g/dl, then at least onehEPO-GMMO may be removed or inactivated to reduce the EPO U by about25%.

In one embodiment, the formulations and method of the present inventionmay be administered with other treatments that are effective in treatinganemia. In one embodiment, other treatments include iron supplements,vitamin B 12 supplements, additional sources of erythropoietin,androgens, growth factors such as G-CSF, or a combination thereof. Inanother embodiment, the formulations and method of the present inventionmay be administered in conjunction with other treatments such as bloodand marrow stem cell transplants.

In one embodiment, this invention provides a therapeutic formulation asdescribed hereinabove in which the therapeutic polypeptide is interferonor in which the therapeutic nucleic acid encodes interferon, which inone embodiment, is interferon alpha, which in one embodiment, isinterferon alpha 2a. In another embodiment, the present inventionprovides a long-lasting interferon-alpha formulation comprising agenetically modified micro-organ, said micro-organ comprising a vectorcomprising a nucleic acid sequence operably linked to one or moreregulatory sequences, wherein said nucleic acid sequence encodesinterferon-alpha and whereby said formulation increases interferon-alphalevels by more than 5% over basal levels and said increasedinterferon-alpha levels persist for greater than one month. In anotherembodiment, the invention provides a method of providing a therapeuticformulation to a subject in need in which the therapeutic polypeptide isinterferon, or in which the therapeutic nucleic acid encodes,interferon, which in one embodiment, is interferon alpha, which in oneembodiment, is interferon alpha 2a. In another embodiment, the inventionprovides a method of providing a therapeutic polypeptide which isinterferon, which in one embodiment, is interferon alpha, which in oneembodiment, is interferon alpha 2a to a subject in need.

In one embodiment, interferons are multi-functional cytokines that arecapable of producing pleitrophic effects on cells, such as anti-viral,anti-proliferative and anti-inflammatory effects. Because of thesecellular responses to interferons, interferon-alpha and interferon-betahave been found to be clinically useful in the treatment of viral,proliferative and inflammatory diseases such as multiple sclerosis,hepatitis B, hepatitis C and several forms of cancer.

Interferon therapies may also have potential use for the treatment ofother inflammatory diseases, viral diseases and proliferative diseases.Thus, a subject in need of interferons may have one or all of theabove-mentioned diseases or conditions.

There are three major classes of interferons: alpha (α), beta (β), andgamma (γ). Aside from their antiviral and anti-oncogenic properties,interferons activate macrophage and natural killer lymphocyte, andenhance major histocompatibility complex glycoprotein classes I and II.Interferon-α is secreted by leukocytes (B-cells and T-cells).Interferon-β is secreted by fibroblasts, and interferon-γ is secreted byT-cells and natural killer lymphocytes.

In one embodiment, the therapeutic polypeptide is interferon alpha, inanother embodiment, interferon beta, or in another embodiment,interferon gamma. In another embodiment, the therapeutic polypeptide isany subtype of interferon alpha, including but not limited to: 1, 2, 4,5, 6, 7, 8, 10, 13, 14, 16, 17, or 21. In another embodiment, thetherapeutic polypeptide is interferon omega, epsilon, kappa, or ahomolog thereof. In another embodiment, the therapeutic polypeptide isinterferon lambda or a homolog thereof. In another embodiment, thetherapeutic polypeptide is any subtype of interferon lambda includingbut not limited to: Interleukin (IL) 28A, IL28B, or IL29. In anotherembodiment, the therapeutic polypeptide is interferon zeta, nu, tau,delta, or a homolog thereof.

In one embodiment, IFNs bind to a specific cell surface receptorcomplex, which in one embodiment is interferon alpha receptor (IFNAR)comprising IFNAR1 and IFNAR2 chains, in another embodiment is interferongamma receptor (IFNGR) complex, which comprises two IFNGR1 and twoIFNGR2 subunits, in another embodiment is a receptor complex comprisingIL10R2 and IFNLR1. In one embodiment, interferons signal through theJAK-STAT signaling pathway.

In one embodiment, the interferon of the formulations and methods of theinstant invention are interferon alpha. In another embodiment, theinterferon of the formulations and methods of the instant invention areinterferon alpha2b. In one embodiment, IFN-alpha-2b is a recombinant,non-glycosylated 165-amino acid alpha interferon protein comprising thegene for IFN-alpha-2b from human leukocytes. IFN-alpha-2b is a type I,water-soluble interferon with a molecular weight of 19,271 daltons(19.271 kDa). In one embodiment, IFN-alpha-2b has a specific activity ofabout 2.6×108 (260 million) International Units/mg as measured by HPLCassay.

In one embodiment, IFN-alpha-2b is one of the Type I interferons, whichbelong to the larger helical cytokine superfamily, which includes growthhormones, interleukins, several colony-stimulating factors and severalother regulatory molecules. All function as regulators of cellularactivity by interacting with cell-surface receptor complexes, known asIFNAR1 and IFNAR2, and activating various signaling pathways.Interferons produce antiviral and anti-proliferative responses in cells.

In one embodiment, a long-lasting IFN-alpha formulation of the presentinvention may be used for the prevention or treatment of hairy cellleukemia, venereal warts, Kaposi's Sarcoma, chronic non-A, non-Bhepatitis, hepatitis B, or a combination thereof. In another embodiment,a long-lasting IFN-alpha formulation of the present invention may beadministered to a subject that is susceptible to one of theabove-mentioned diseases or conditions or has been or will be exposed toan infectious agent, as described herein. In another embodiment, along-lasting IFN-alpha formulation invention may be used for theprevention or treatment of hepatitis C. According to this aspect and inone embodiment, the formulations of the present invention may beadministered concurrently or alternately with other hepatitis Ctreatments, including inter alia, ribavarin, interferons, pegylatedinterferons or a combination thereof.

In another embodiment, a long-lasting IFN-alpha formulation may be usedor evaluated alone or in conjunction with chemotherapeutic agents in avariety of other cellular proliferation disorders, including chronicmyelogenous leukemia, multiple myeloma, superficial bladder cancer, skincancers (including, inter alia, basal cell carcinoma and malignantmelanoma), renal cell carcinoma, ovarian cancer, low grade lymphocyticand cutaneous T cell lymphoma, and glioma. In another embodiment, along-lasting IFN-alpha formulation may be used for the prevention ortreatment of solid tumors that arise from lung, colorectal and breastcancer, alone or with other chemotherapeutic agents. In anotherembodiment, a long-lasting IFN-alpha formulation may be used for theprevention or treatment of multiple sclerosis. In another embodiment, along-lasting IFN-alpha formulation may be used for the prevention ortreatment of histiocytic diseases, which in one embodiment isErdheim-Chester disease (ECD), which in one embodiment is a potentiallyfatal disorder that attacks the body's connective tissue and in oneembodiment is caused by the overproduction of histiocytes, which in oneembodiment, accumulate in loose connective tissue, causing it to becomethickened and dense. In another embodiment, a long-lasting IFN-alphaformulation may be used for the prevention or treatment of severe ocularBehcet's disease.

In one embodiment, the interferon alpha gene has a nucleic acid sequencethat corresponds to that set forth in Genbank Accession Nos: K01900;M11003; or M71246, or encodes a protein sequence that corresponds tothat set forth in Genbank Accession Nos: AAA52716; AAA52724; orAAA52713. In one embodiment, the interferon beta gene has a nucleic acidsequence that corresponds to that set forth in Genbank Accession Nos:M25460; AL390882; or CH236948, or encodes a protein sequence thatcorresponds to that set forth in Genbank Accession Nos: AAC41702;CAH70160; or EAL24265. In one embodiment, the interferon gamma gene hasa nucleic acid sequence that corresponds to that set forth in GenbankAccession Nos: J00219; AF506749; NM_(—)000619; or X62468, or encodes aprotein sequence that corresponds to that set forth in Genbank AccessionNos: AAB59534; AAM28885; NP_(—)000610; or CAA44325. In anotherembodiment, the interferon alpha gene has a nucleic acid sequence aspresented in SEQ ID No: 8, while in another embodiment, the interferonalpha gene has an amino acid sequence as presented in SEQ ID No: 9. Inanother embodiment, the interferon alpha gene has a nucleic acid that ishomologous to that presented in SEQ ID No: 8, while in anotherembodiment, the interferon alpha gene has an amino acid sequence that ishomologous to that presented in SEQ ID No: 9.

In another embodiment, the present invention provides a method ofdelivering interferon-alpha to a subject in need over a sustained periodcomprising: providing one or more genetically modified micro-organs,said micro-organs comprising a vector comprising a nucleic acid sequenceoperably linked to one or more regulatory sequences; and implanting saidgenetically modified micro-organ in said subject, wherein said nucleicacid sequence encodes interferon-alpha and whereby interferon-alphalevels are increased by more than 5% over basal levels and saidincreased interferon-alpha levels persist for greater than one month.

In one embodiment, the formulations and methods of the present inventionprovide a nucleic acid optimized for increased expression levels,duration, or a combination thereof of a therapeutic polypeptide encodedby said nucleic acid. In another embodiment, the invention provides anucleic acid sequence with greater than 85% homology to SEQ ID No: 1, avector comprising such a nucleic acid sequence, and a cell comprisingsuch as vector.

In another embodiment, the invention provides a nucleic acid sequencewith greater than 85% homology to SEQ ID No: 2, a vector comprising sucha nucleic acid sequence, and a cell comprising such as vector.

The term “homology”, as used herein, when in reference to any nucleicacid sequence indicates a percentage of nucleotides in a candidatesequence that is identical with the nucleotides of a correspondingnative nucleic acid sequence.

In one embodiment, the terms “homology”, “homologue” or “homologous”, inany instance, indicate that the sequence referred to, exhibits, in oneembodiment at least 70% correspondence with the indicated sequence. Inanother embodiment, the nucleic acid sequence exhibits at least 72%correspondence with the indicated sequence. In another embodiment, thenucleic acid sequence exhibits at least 75% correspondence with theindicated sequence. In another embodiment, the nucleic acid sequenceexhibits at least 77% correspondence with the indicated sequence. Inanother embodiment, the nucleic acid sequence exhibits at least 80%correspondence with the indicated sequence. In another embodiment, thenucleic acid sequence exhibits at least 82% correspondence with theindicated sequence. In another embodiment, the nucleic acid sequenceexhibits at least 85% correspondence with the indicated sequence. Inanother embodiment, the nucleic acid sequence exhibits at least 87%correspondence with the indicated sequence. In another embodiment, thenucleic acid sequence exhibits at least 90% correspondence with theindicated sequence. In another embodiment, the nucleic acid sequenceexhibits at least 92% correspondence with the indicated sequence. Inanother embodiment, the nucleic acid sequence exhibits at least 95% ormore correspondence with the indicated sequence. In another embodiment,the nucleic acid sequence exhibits 95%-100% correspondence to theindicated sequence. Similarly, reference to a correspondence to aparticular sequence includes both direct correspondence, as well ashomology to that sequence as herein defined.

Homology may be determined by computer algorithm for sequence alignment,by methods well described in the art. For example, computer algorithmanalysis of nucleic acid sequence homology may include the utilizationof any number of software packages available, such as, for example, theBLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT andTREMBL packages.

An additional means of determining homology is via determination ofnucleic acid sequence hybridization, methods of which are well describedin the art (See, for example, “Nucleic Acid Hybridization” Hames, B. D.,and Higgins S. J., Eds. (1985); Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, (Volumes 1-3) Cold Spring Harbor Press,N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology,Green Publishing Associates and Wiley Interscience, N.Y). In oneembodiment, methods of hybridization may be carried out under moderateto stringent conditions. Hybridization conditions being, for example,overnight incubation at 42° C. in a solution comprising: 10-20%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20μg/ml denatured, sheared salmon sperm DNA.

In one embodiment, the present invention provides therapeuticformulations comprising micro-organs and methods of use thereof. In oneembodiment, the preparation of therapeutic micro-organs comprises (a)obtaining a plurality of micro-organ explants from a donor subject, eachof the plurality of micro-organ explants comprises a population ofcells, each of the plurality of micro-organ explants maintaining amicroarchitecture of an organ from which it is derived and at the sametime having dimensions selected so as to allow diffusion of adequatenutrients and gases to cells in the micro-organ explants and diffusionof cellular waste out of the micro-organ explants so as to minimizecellular toxicity and concomitant death due to insufficient nutritionand accumulation of the waste in the micro-organ explants; (b)genetically modifying the plurality of micro-organ explants, so as toobtain a plurality of genetically modified micro-organ explants, saidmicro-organs comprising and secreting the proteins differing by the atleast one amino acid; and (c) implanting the plurality of geneticallymodified micro-organ explants within a plurality of recipient subjects.

Methods for the preparation and processing of micro-organs intogenetically modified micro-organs are disclosed in WO2004/099363,incorporated herein by reference in their entirety. Micro-organscomprise tissue dimensions defined such that diffusion of nutrients andgases into every cell in the three dimensional micro-organ, andsufficient diffusion of cellular wastes out of the explant, is assured.Ex vivo maintenance of the micro-organs, which in one embodiment, is inminimal media, can continue for an extended period of time, whereuponcontrolled ex vivo transduction incorporating desired gene candidateswithin cells of the micro-organs using viral or non-viral vectorsoccurs, thus creating genetically modified micro-organs.

In one embodiment, micro-organs are harvested using a drill and coringneedle, as described hereinbelow. In another embodiment, micro-organsare harvested using a harvesting system that utilizes a vacuum to holdthe skin taut and open the slits during insertion of the coring drill.In another embodiment, any tool which may be used to harvest dermaltissue may be used to harvest micro-organs of the appropriate size,including but not limited to those tools and methods described in PCTApplication WO 04/099363.

Incorporation of recombinant nucleic acid within the micro-organs togenerate genetically modified micro-organs or biopumps can beaccomplished through a number of methods well known in the art. Nucleicacid constructs can be utilized to stably or transiently transduce themicro-organ cells. In stable transduction, the nucleic acid molecule isintegrated into the micro-organ cells genome and as such it represents astable and inherited trait. In transient transduction, the nucleic acidmolecule is maintained in the transduced cells as an episome and isexpressed by the cells but it is not integrated into the genome. Such anepisome can lead to transient expression when the transduced cells arerapidly dividing cells due to loss of the episome or to long termexpression wherein the transduced cells are non-dividing cells.

Typically the nucleic acid sequence is subcloned within a particularvector, depending upon the preferred method of introduction of thesequence to within the micro-organs, as described hereinabove. Once thedesired nucleic acid segment is subcloned into a particular vector itthereby becomes a recombinant vector.

In one embodiment, micro-organs are incubated at 32° C. before and aftergenetic modification, while in another embodiment, they are incubated at37° C. In another embodiment, micro-organs are incubated at 33° C., 34°C., 35° C., 36° C., 38° C., 39° C., 40° C., 28° C., 30° C., 31° C., 25°C., 42° C., or 45° C.

In one embodiment, micro-organs are incubated at 10% CO₂ before andafter genetic modification, while in another embodiment, they areincubated at 5% CO₂. In another embodiment, micro-organs are incubatedat 12% CO₂, 15% CO₂, 17% CO₂, or 20% CO₂. In another embodiment,micro-organs are incubated at 2% CO₂, 6% CO₂, 7% CO₂, 8% CO₂, or 9% CO₂.

In another embodiment, incubation temperatures, CO₂ concentrations, or acombination thereof may be kept at a single temperature or concentrationbefore, during, and after genetic modification, while in anotherembodiment, incubation temperatures, CO₂ concentrations, or acombination thereof may be adjusted at different points before, during,and after genetic modification of micro-organs.

In another embodiment, micro-organs are incubated at 85-100% humidity,which in one embodiment is 95% humidity, in another embodiment, 90%humidity, and in another embodiment, 98% humidity.

In one embodiment, the levels of therapeutic nucleic acids orpolypeptides may be detected using any method known in the art. Theefficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art. For example, DNA introduced into acell can be detected by a filter hybridization technique (e.g., Southernblotting) and RNA produced by transcription of introduced DNA can bedetected, for example, by Northern blotting, RNase protection or reversetranscriptase-polymerase chain reaction (RT-PCR). The gene product canbe detected by an appropriate assay, for example by immunologicaldetection of a produced protein, such as with a specific antibody, or bya functional assay to detect a functional activity of the gene product,such as an enzymatic assay. In one embodiment, ELISA, Western blots, orradioimmunoassay may be used to detect proteins. If the gene product ofinterest to be expressed by a cell is not readily assayable, anexpression system can first be optimized using a reporter gene linked tothe regulatory elements and vector to be used. The reporter gene encodesa gene product which is easily detectable and, thus, can be used toevaluate efficacy of the system. Standard reporter genes used in the artinclude genes encoding β-galactosidase, chloramphenicol acetyltransferase, luciferase and human growth hormone.

Thus, in one embodiment, therapeutic polypeptide or nucleic acidexpression levels are measured in vitro, while in another embodiment,therapeutic polypeptide or nucleic acid expression levels are measuredin vivo. In one embodiment, in vitro determination of polypeptide ornucleic acid expression levels, which in one embodiment, is EPO levelsand in another embodiment, IFN-alpha levels, allows a determination ofthe number of micro organs to be implanted in a patient via determiningthe secretion level of a therapeutic agent by a micro-organ in vitro;estimating a relationship between in vitro production and secretionslevels and in vivo serum levels of the therapeutic agent; anddetermining an amount of the therapeutic formulation to be implanted,based on the determined secretion level and the estimated relationship.

In another preferred embodiment of this invention, polynucleotide(s) canalso include trans-, or cis-acting enhancer or suppresser elements whichregulate either the transcription or translation of endogenous genesexpressed within the cells of the micro-organs, or additionalrecombinant genes introduced into the micro-organs. Numerous examples ofsuitable translational or transcriptional regulatory elements, which canbe utilized in mammalian cells, are known in the art.

For example, transcriptional regulatory elements comprise cis- ortrans-acting elements, which are necessary for activation oftranscription from specific promoters [(Carey et al., (1989), J. Mol.Biol. 209:423-432; Cress et al., (1991), Science 251:87-90; and Sadowskiet al., (1988), Nature 335:5631-564)].

Translational activators are exemplified by the cauliflower mosaic virustranslational activator (TAV) [see for example, Futterer and Hohn,(1991), EMBO J. 10:3887-3896]. In this system a bi-cistronic mRNA isproduced. That is, two coding regions are transcribed in the same mRNAfrom the same promoter. In the absence of TAV, only the first cistron istranslated by the ribosomes, however, in cells expressing TAV, bothcistrons are translated.

The polynucleotide sequence of cis-acting regulatory elements can beintroduced into cells of micro-organs via commonly practiced geneknock-in techniques. For a review of gene knock-in/out methodology see,for example, U.S. Pat. Nos. 5,487,992, 5,464,764, 5,387,742, 5,360,735,5,347,075, 5,298,422, 5,288,846, 5,221,778, 5,175,385, 5,175,384,5,175,383, 4,736,866 as well as Burke and Olson, Methods in Enzymology,194:251-270, 1991; Capecchi, Science 244:1288-1292, 1989; Davies et al.,Nucleic Acids Research, 20 (11) 2693-2698, 1992; Dickinson et al., HumanMolecular Genetics, 2(8):1299-1302, 1993; Duff and Lincoln, “Insertionof a pathogenic mutation into a yeast artificial chromosome containingthe human APP gene and expression in ES cells”, Research Advances inAlzheimer's Disease and Related Disorders, 1995; Huxley et al.,Genomics, 9:742-750 1991; Jakobovits et al., Nature, 362:255-261 1993;Lamb et al., Nature Genetics, 5: 22-29, 1993; Pearson and Choi, Proc.Natl. Acad. Sci. USA, 1993, 90:10578-82; Rothstein, Methods inEnzymology, 194:281-301, 1991; Schedl et al., Nature, 362: 258-261,1993; Strauss et al., Science, 259:1904-1907, 1993, WO 94/23049, WO93/14200, WO 94/06908 and WO 94/28123 also provide information.

Down-regulation of endogenous sequences may also be desired, in order toassess production of the recombinant product exclusively. Toward thisend, antisense RNA may be employed as a means of endogenous sequenceinactivation. Exogenous polynucleotide(s) encoding sequencescomplementary to the endogenous mRNA sequences are transcribed withinthe cells of the micro-organ. Down regulation can also be effected viagene knock-out techniques, practices well known in the art (“MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988)).

Over expression of the recombinant product may be desired as well. Overexpression may be accomplished by providing a high copy number of one ormore coding sequences in the respective vectors. These exogenouspolynucleotide sequences can be placed under transcriptional control ofa suitable promoter of a mammalian expression vectors to regulate theirexpression. In another embodiment, multiple copies of the same gene orof several related genes may be used as a means to increase polypeptideor nucleic acid expression. In one embodiment, expression is stabilizedby DNA elements, which in one embodiment are matrix-associating regions(MARs) or scaffold-associating regions (SARs).

In one embodiment, an adenoviral vector is the vector of thecompositions and for use in the methods of the present invention. In anembodiment in which an adenoviral vector is used as a vector, thehelper-dependent adenovirus system may be used in one embodiment, toprepare therapeutic polypeptide or nucleic acid-expressinghelper-dependent adenovirus vector for transforming micro-organs. In oneembodiment, such a helper-dependent adenovirus system comprises ahelper-dependent adenovirus, a helper virus, and a producer cell line isused in the preparation of the formulation of the present invention isas described in Palmer and Ng, 2003 Mol Ther 8:846 and in Palmer and Ng,2004 Mol Ther 10:792, which are hereby incorporated herein by referencein their entirety.

In one embodiment, a helper cell line, designated 293, which wastransformed from human embryonic kidney cells by Ad5 DNA fragments andconstitutively expresses E1 proteins is used to generate and propagatereplication deficient adenoviral vectors. In another embodiment, helpercell lines may be derived from human muscle cells, hematopoietic cellsor other human embryonic mesenchymal or epithelial cells. Alternatively,the helper cells may be derived from the cells of other mammalianspecies that are permissive for human adenovirus. Such cells include,e.g., Vero cells or other monkey embryonic mesenchymal or epithelialcells.

In one embodiment, micro-organs are maintained ex vivo for a period oftime, which may range from several hours to several months. In oneembodiment, maintenance ex vivo refers to maintenance of a micro-organfollowing ex vivo genetic manipulation using a viral vector, i.e.,maintenance of a GMMO. In another embodiment, maintenance ex vivo refersto maintenance of a micro-organ prior to genetic manipulation thereof.

In one embodiment, genetically modified micro-organs are maintained forseveral days, and in another embodiment, for several weeks prior toimplantation. In one embodiment, micro-organs are maintained for between9-14 days prior to implantation. In one embodiment, micro-organs aremaintained for between two to four weeks prior to implantation. In oneembodiment, micro-organs are maintained for three weeks prior toimplantation. In one embodiment, micro-organs are maintained for fourweeks or more prior to implantation. In yet another embodiment,micro-organs are maintained for at least 9 days.

Without being limited by theory, in one embodiment, said incubationallows cells to process and break down viral proteins, which in oneembodiment are viral capsids, present as a result of viral vectortransduction. In one embodiment, such a turnover of capsid proteinsoccurs within 2-3 days, so that, in one embodiment, little if any viralcapsid proteins remain by the 10^(th) day ex vivo. In one embodiment,the breaking down of viral capsids further reduces the immunogenicity ofthe formulations of the instant invention and increases the expressionlevels and expression duration of the gene or genes of interest. Inanother embodiment, said incubation allows the early HD-Advector-induced innate immune responses to occur in vitro, which in oneembodiment, will not persist beyond 24 hours in the absence of Adenogene transcription. In another embodiment, the later adaptive responsesthat normally follow the administration of transcription-competentfirst-generation-Ad vectors, which are predominantly characterized inone embodiment, by lymphocyte infiltration and in another embodiment byinduction of Ad-specific CTL's, are not be elicited by HD-Ad vectors.

In one embodiment, the ex vivo micro-organ is exposed to viral vector ata dosage of 1.6-3×10⁹ infectious particles (ip)/ml, 3-4×10¹² viralparticles/ml, or 2×10¹¹ viral particles/ml. In another embodiment, exvivo micro-organs are exposed to viral vector at a dosage of 1×10³ to1×10¹² viral particles/ml, in another embodiment from 1×10³ to 1×10⁹,and in another embodiment, from 1×10⁶ to 1×10⁹ and in anotherembodiment, 1×10⁶ to 1×10¹² viral particles/ml. In one embodiment, thedosage of viral particles/g body weight of subject that are administeredto a subject within a micro-organ is less than 1×10³, and in anotherembodiment, less than 1×10², and in another embodiment, less than 1×10¹viral particles/g body weight of subject.

In one embodiment, growth factors are used to increase the number ofcells in the micro-organs.

In one embodiment, in vitro expression can be assessed prior toimplantation, enabling the possibility for in vitro to in vivocorrelation studies of expressed recombinant proteins.

In some embodiments of the invention, the amounts of tissue sampleincluding a genetically modified cell(s) to be implanted are determinedfrom one or more of: corresponding amounts of the therapeutic agent ofinterest routinely administered to such subjects based on regulatoryguidelines, specific clinical protocols or population statistics forsimilar subjects, corresponding amounts of the therapeutic agent such asprotein of interest specifically to that same subject in the case thathe/she has received it via injections or other routes previously,subject data such as weight, age, physical condition, clinical status,pharmacokinetic data from previous tissue sample which includes agenetically modified cell administration to other similar subjects,response to previous tissue sample which includes a genetically modifiedcell administration to that subject, or a combination thereof. Thus, inone embodiment, the level of expression of gene products by one or moremicro-organs is determined in vitro, a relationship between in vitro andin vivo therapeutic polypeptide or nucleic acid expression levels isdetermined or estimated, and the number of micro-organs to be implantedin a particular patient is determined based on the calculated orestimated relationship. The dosage of the therapeutic agent may beadjusted as described previously (WO2004/099363).

In one embodiment, a micro-organ or a genetically modified micro-organmay be maintained in vitro for a proscribed period of time until theyare needed for implantation into a host. In one embodiment, amicro-organ or a genetically modified micro-organ may be maintained orstored in culture for between 1-7 days, between 1-8 weeks, or for 1-4months. In another embodiment, the therapeutic agent, left in thesupernatant medium surrounding the tissue sample, can be isolated andinjected or applied to the same or a different subject.

Alternatively or additionally, a genetically modified micro-organ can becryogenically preserved by methods known in the art, for example,without limitation, gradual freezing (0° C., −20° C., −80° C., −196° C.)in DMEM containing 10% DMSO, immediately after being formed from thetissue sample or after genetic alteration.

Administration of the formulation of the invention may be by implantinginto the subject in need. In one embodiment, the formulation of theinstant invention may be implanted in an organ or system that isaffected by a disease or disorder to be treated or prevented by a methodor route which results in localization of the micro-organ at a desiredsite. In another embodiment, the location of the implanted formulationmay be distal from an organ or system that is affected by a disease ordisorder. Thus, while in one embodiment, the recombinant protein isreleased locally, in another embodiment, the recombinant proteindiffuses to the lymphatic system, which in one embodiment, mayultimately lead to systemic distribution of the recombinant protein.Thus, the present invention provides for the use of therapeuticformulations in various concentrations to treat a disease or disordermanifesting in any part of the subject in need.

According to this aspect and in one embodiment, formulations of theinstant invention may be implanted intratumorally. In anotherembodiment, formulations may be implanted at a site distal from thetumor, which in one embodiment is associated with metastasis of aparticular type of tumor. In another embodiment, formulations of theinstant invention may be implanted into the kidney of a subject, whichin one embodiment is a subcapsular implantation. In another embodiment,formulations of the instant invention are implanted laparascopically.

In one embodiment, the formulations of the invention may be implanted asingle time for acute treatment of temporary conditions, or may beimplanted more than one time, especially in the case of progressive,recurrent, or degenerative disease. In one embodiment, one or moreformulations of the invention may be administered simultaneously, or inanother embodiment, they may be administered in a staggered fashion. Inone embodiment, the staggered fashion may be dictated by the stage orphase of the disease.

In one embodiment, the micro-organ is implanted at a desired location inthe subject in such a way that at least a portion of the cells of themicro-organ remain viable. In one embodiment of this invention, at leastabout 5%, in another embodiment of this invention, at least about 10%,in another embodiment of this invention, at least about 20%, in anotherembodiment of this invention, at least about 30%, in another embodimentof this invention, at least about 40%, and in another embodiment of thisinvention, at least about 50% or more of the cells remain viable afteradministration to a subject. The period of viability of the cells afteradministration to a subject can be as short as a few hours, e.g.,twenty-four hours, to a few days, to as long as a few weeks to months oryears.

Micro-organ implantation within a recipient subject provides for asustained dosage of the recombinant product. The micro-organs may beprepared, prior to implantation, for efficient incorporation within thehost facilitating, for example, formation of blood vessels within theimplanted tissue. Recombinant products may therefore be deliveredimmediately to peripheral recipient circulation, following production.Alternatively, micro-organs may be prepared, prior to implantation, toprevent cell adherence and efficient incorporation within the host.Examples of methods that prevent blood vessel formation includeencasement of the micro-organs within commercially availablecell-impermeant diameter restricted biological mesh bags made of silk ornylon, or others such as, for example GORE-TEX bags (Terrill P J,Kedwards S M, and Lawrence J C. (1991) The use of GORE-TEX bags for handburns. Burns 17(2): 161-5), or other porous membranes that are coatedwith a material that prevents cellular adhesion, for example Teflon.

Gene products produced by micro-organs can then be delivered via, forexample, polymeric devices designed for the controlled deliverycompounds, e.g., drugs, including proteinaceous biopharmaceuticals. Avariety of biocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a gene product of the micro-organsin context of the invention at a particular target site. The generationof such implants is generally known in the art (see, for example,Concise Encyclopedia of Medical & Dental Materials, ed. By DavidWilliams (MIT Press: Cambridge, Mass., 1990); Sabel et al. U.S. Pat. No.4,883,666; Aebischer et al. U.S. Pat. No. 4,892,538; Aebischer et al.U.S. Pat. No. 5,106,627; Lim U.S. Pat. No. 4,391,909; and Sefton U.S.Pat. No. 4,353,888). In one embodiment, a GMMO is encapsulated. Inanother embodiment, a GMMO is not encapsulated.

Implantation of genetically modified micro-organs according to thepresent invention can be effected via standard surgical techniques orvia injecting micro-organ preparations into the intended tissue regionsof the mammal utilizing specially adapted syringes employing a needle ofa gauge suitable for the administration of micro-organs. In anotherembodiment, a catheter is employed for implanted micro-organs. In oneembodiment, any of the implantation methods described in PCT PublicationWO2 04/099363 may be used and is considered an embodiment of thisinvention.

In one embodiment, micro-organs are implanted subcutaneously,intradermally, subdermally, intramuscularly, intraperitoneally orintragastrically. In one embodiment, the term implanted excludes beinggrafted as a split-thickness or full-thickness skin graft. In oneembodiment of the present invention, the donor micro-organs utilized forimplantation are preferably prepared from an organ tissue of therecipient mammal (i.e. autologous), or a syngeneic mammal, althoughallogeneic and xenogeneic tissue can also be utilized for thepreparation of the micro-organs providing measures are taken prior to,or during implantation, so as to avoid graft rejection and/or graftversus host disease (GVHD). As used herein, GVHD refers to graft versushost disease, a consequence of tissue transplantation (the graft) causedby the transplant immune response against the recipient host. Morespecifically, graft-versus-host disease is caused by donor T-lymphocytes(T cells), recognizing the recipient as being foreign and attackingcells of the recipient. Numerous methods for preventing or alleviatinggraft rejection or GVHD are known in the art and may be used in themethods of this invention. In one embodiment, to facilitatetransplantation of the cell populations within a tissue which may besubject to immunological attack by the host, e.g., where xenogenicgrafting is used, such as swine-human transplantations, the micro-organmay be inserted into or encapsulated by biocompatible immuno-protectedmaterial such as rechargeable, non-biodegradable or biodegradabledevices and then transplanted into the recipient subject. In anotherembodiment, the micro-organ is not inserted into or encapsulated bybiocompatible immuno-protected material such as rechargeable,non-biodegradable or biodegradable devices.

In another embodiment, the donor micro-organs utilized for implantationare preferably prepared from a donor who is human leukocyte antigen(HLA)-matched with the recipient, where in one embodiment, HLA is themajor histocompatibility complex in humans. In one embodiment, donor andrecipient are matched for class I major histocompatibility complex (MHC)genes, class II MHC genes, or a combination thereof. In one embodiment,class I MHC genes comprise HLA-A, HLA-B, and HLA-C, wherein in oneembodiment, a mismatch of class I MHC genes increases the risk of graftrejection, and in one embodiment, class II MHC genes comprise HLA-DPA1,HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, wherein in oneembodiment, a mismatch of class II MHC genes increases the risk of GVHD.In another embodiment, donor and recipient are matched for HLA-DM andHLA-DO genes.

In one embodiment, viral turnover or elimination from cells ex vivo isenhanced via techniques know in the art, such as physical methods, whichin one embodiment is heating, use of antiviral agents, agents whichstimulate viral turnovers by cells, etc.

In one embodiment, while the long-lasting formulations of the presentinvention increase the level and duration of nucleic acid or polypeptideexpression, the levels of nucleic acid or polypeptide expression do notremain elevated indefinitely.

In contrast to other methods involving transient transduction of cells,or cells that turn over rapidly, the long-lasting EPO formulation of theinstant invention comprises cells that are no longer replicating.Therefore, the EPO formulation produces a stable protein from a stableconstruct and is expected to continue producing the protein alreadycharacterized.

EXAMPLES Experimental Materials and Methods

Materials and Equipment List

Production medium was used to grow micro-organs and comprises DMEM-HEPESMedium (High glucose 4,500 mg/L and 25 mM HEPES; Hi-CloneCat#SH3A1448.02) comprising 1% glutamine and supplemented with 50 μg/mlGentamycin (RAFA labs, for injection) and 0.1% Amphotericin B (BMS,Fungizone I.V.) (final concentration in the media 2.5 μg/ml AmphotericinB). In some experiments, 10% serum substitute supplement (SSS, IrvineScientific, Cat #99193), 10% autologous human serum, or 10% Fetal bovineserum (FBS or FCS) was added to the production medium.

Harvesting of Dermal Micro-Organs

A. Method I.

Human dermal micro-organs were harvested from an area of skin from aregion of the donor's lower abdomen. In certain instances, thedimensions of the dermal micro-organs harvested were approximately1.5-2.5 mm in width and 30 mm in length. In some cases, to prevent theharvest of the epidermis, a shallow slit (1-2 mm deep) passing throughthe stratum cornea into the dermis was cut along a straight line at oneside of the skin region from which the micro-organs were to beharvested, and a similar slit was cut 30 mm away from and parallel tothe first slit. The distance between the slits determined themicro-organ length and was consistent throughout the experiments.

In certain cases, a thin gauge (typically 22GA) hypodermic needleattached to a 1 ml syringe filled with sterile saline was inserted intothe exposed dermis at the first slit and slid along the dermis of theharvesting site towards the opposite slit, with the needles angled asnecessary so that it exited through the dermis at the opposite slit.

Next, in some cases, the outer skin along the length of the guidingneedle was pinched with a surgical clamp. The needle embedded in thedermis was lifted slightly to raise the area of skin surrounding it andsometimes a hook shaped device beneath the inserted hypodermic needle'spoint was used to assist in lifting the skin before it's pinched. Thetip of the guide needle protruding from its point of exit, was insertedinto the sharp leading end of a coring needle (1-3 mm in diameter, PointTechnologies, Colo. USA), which was held by a commercially availabledrill (such as Aesculap Micro Speed GD 650, GD 657). A small amount ofsterile saline was injected from the syringe into the coring needle. Thedrill is activated to rotate the coring needle at high speed (typically3000-7000 RPM) and while rotating, the drill and coring needle aremanually urged forward along the axis of the guide needle to cut a 30-40mm long cylindrical dermal core (dermal micro-organ) having an outerdiameter approximately that of the inner diameter of the coring needle.The dermal micro-organ usually remained attached to the guide needle,which was withdrawn from within the coring needle and placed inProduction media (as described hereinabove), and the coring needle wasremoved from the skin.

In many cases, using tweezers, each micro-organ was transferred to alabeled single well in a 24 well plate containing 1000 μl ProductionMedium. To remove the debris, two additional media changes of 1000 μlwere performed for each micro-organ. The plates containing themicro-organs in 1000 μl production media were then transferred to anincubator that had been equilibrated to 32° C., 10% CO₂, and ˜95%humidity for a 24 hr recovery period.

B. Method II.

Micro-organs (MOs) are harvested under local anesthesia from the dermisof the lower abdomen or the upper or lower back of the patient that willbe treated, with dimensions of approximately 1.5-2.5 mm in width, and 30mm in length. Their dimensions and appearance remain essentially thesame during the entire hEPO-GMMO Biopump ex-vivo production process.

The dermis micro-organs are transported under controlled conditions to acontract cGMP (current Good Manufacturing Practice) processing facility.Upon receipt at the cGMP processing center, the MOs are divided into twosubsets: those to be processed immediately into Biopumps, and those thatwill be cryopreserved for later processing, if required, into Biopumps.The decision as to how many will be processed immediately will be basedon an estimate of the EPO dose needed for that patient and an estimateof average secretion levels from typical Biopumps based on thousands ofprocessed Biopumps in pre-clinical and clinical testing.

For the MOs that are processed immediately, the HDAd-EPO vector is usedto perform the transduction. After transduction, residual viralparticles are removed by several media exchanges. The Biopumps(transduced micro-organs) are maintained in culture for approximatelyone week in order to assay for protein secretion levels and sterility.Release criteria of processed Biopumps are listed in Table 1.

TABLE 1 Test Sampled Release Criteria Sterility Day of release No growthof bacteria or fungi at 14 days. Gram Stain Day of release NegativeMycoplasma 3 days prior to release Negative Endotoxin 3 days prior torelease Less than 0.5 EU/mL EPO identity 3 days prior to release andPositive for EPO day of release secretion EPO activity 3 days prior torelease and EPO secretion >100 U/ day of release Biopump/day Glucose 3days prior to release and Total glucose consumption day of releaseconsumed >150 μg/ Biopump/3 days

The Biopumps are then transported under controlled conditions back tothe treatment center for subcutaneous implantation under localanesthesia in the abdominal wall or the upper or lower back of the samepatient.

Micro-organs retrieved from cryostorage for processing, if required,into Biopumps are first thawed prior to undergoing the transduction andmaintenance procedures, as detailed above. Data demonstrate thecomparability of Biopumps produced from fresh and cryopreserved MOs.

The decision as to how many dermal micro-organs are transduced at anygiven time, is based upon the estimated dose needed for the patient andan estimate of average secretion levels from typical geneticallymodified dermal micro-organs. Dermal micro-organs not used immediatelyfor transduction are cryopreserved for later processing

Virus Transduction

Each micro-organ was transferred for transduction into a well of a48-well plate, which have smaller wells requiring smaller total fluidvolume, to conserve virus. The medium was carefully removed from eachwell without disturbing the micro-organ inside. During the preclinicalexperiments, three different vectors were tested: 1.6-3×10⁹ infectiousparticles (ip)/ml of first generation adenovirus (Molecular Medicine),approximately 3-4×10¹² viral particles/ml helper-dependent adenovirus(Baylor), or approximately 2×10″ viral particles/ml adeno-associatedvirus (University of Pennsylvania), each comprising recombinant humanEPO gene, optimized recombinant human EPO gene, or optimized IFN-alphagene, were each diluted 1:10, 1:25, 1:50, 1:100, 1:500, or 1:1000 inDMEM-HEPES (Gibco Cat#42430-025) with or without FCS. Each well of the48-well plates was filled with 100 μL of one of the diluted titers of avirus. The plate was placed in a CO₂ incubator and transduction wasassisted by agitation on a digital microtiter shaker at 300 rpm for aperiod of 2 hours and an additional 16-22 hour incubation withoutshaking.

The transduced micro-organs were transferred to a 24-well plate aftertransduction and then washed three times with 1 mL production media(without FCS) to remove the non-transduced viral particles. Afterwashing, the biopumps were maintained in 1 mL production media in astandard high humidity CO₂ incubator at 95% humidity, 10% CO₂, and 32°C. Seventy-two hours after the removal of the viral vector, theproduction medium was replaced with fresh medium, and aliquots of thespent medium were assayed for secreted recombinant protein levels.

HDAd-EPO Vector for Clinical Trials

The delivery vector for the clinical trials described below in Examples3 and 4, which is used ex-vivo to transduce the cells of the micro-organtissue samples which are biopsied from the dermis of the patient beingtreated, is a non-replicating helper-dependent Adenoviral (HDAd) vector.

The HDAd vector may provide sustained protein expression and may preventimmune rejection. HDAd vectors lack all viral protein coding sequencesand contain only the cis-acting elements required to replicate andpackage the vector DNA. Consequently, HDAd vectors may avoid theadaptive immune response that normally follows the administration offirst-generation Adeno vectors, thus giving the HDAd vectors animmunological advantage [Muruve D A, et al, Helper-dependent adenovirusvectors elicit intact innate but attenuated adaptive host immuneresponses in vivo. J Virol 2004; 78 (11):5966-5972. In addition, theresults of Examples 1, 2 and 5 demonstrated that HDAd vectors exhibitstable long-term transgene expression ex vivo. HDAd retain all thefirst-generation adenovirus advantages including high titer production,efficient infection of a broad range of cell types, and the ability toinfect dividing and nondividing cells. Moreover, they express no viralproteins, can accommodate up to 36 kb of foreign DNA, and exhibitreduced tissue toxicity and prolonged transgene expression as comparedwith first-generation adenoviral vectors.

The expression cassette within the HDAd vector includes the gene forerythropoietin and a CAG promoter. FIG. 17 presents a map of theelements of the expression cassette. The nucleic acid sequence of theCAG-EPO expression cassette for Examples 3 and 4 is presented as SEQ IDNo. 11. Included are multiple cloning site sequences (SEQ ID Nos. 14 and15); a CAG promoter sequence (SEQ ID No. 12); human EPO intron-less genefrom ATG to the stop codon (SEQ ID No. 7); and SV40 poly A sequence (SEQID No. 13).

The major steps in the derivation of the HDAd construct were as follows:pAd-CMV-EPO→pAd-CAG-EPO→pΔ28E4-CAG-EPO→HDAd-CAG-EPO.

Construction of pAd-CAG-EPO plasmid was as follows. The pAD-CMV-EPOvector was provided by Dr. Paul Robbins from the University ofPittsburgh (Hardy S., et al., 1997, J. Virol. 71(3):1842-1849, Lippin Y.et al., 2005, Blood 106(7): 2280-2286). In order to achieve higherlevels of transgene expression, traditional cloning techniques have beenused to replace the CMV promoter with the CAG promoter, in order toarrive at the pAd-CAG-EPO plasmid.

Construction of pΔ28E4-CAG-EPO plasmid was as follows: pΔ28E4 (Toiettaet al., 2002, Mol. Ther. 5: 204-210) was used as the backbone to createthe pΔ28E4-CAG-EPO plasmid. This pΔ28E4 plasmid contains two genomichuman DNA stuffer sequences: the first corresponds to chromosome x HPRT1(accession#AC004383 nt 60989-44991), and the second fragment correspondsto chromosome x clone CTD-2537J14 map q28 (accession#AC109994 nt66942-78052). These DNA fragments were inserted into pΔ28E4 as stufferDNA in order to achieve the correct vector size that will enable theefficient generation of HDAd viral particles.

The pΔ28E4-CAG-EPO plasmid was constructed by conventional cloningtechniques. The CAG-EPO expression cassette from pAd-CAG-EPO plasmid wasinserted into the AscI unique site of the HDAd cloning shuttle vector,pΔ28E4, generating the pΔ28E4-CAG-EPO construct, which has theappropriate size for efficient and stable viral packaging. FIG. 18illustrates the cloning steps for the pΔ28E4-CAG-EPO plasmid.

In addition to the HDAd-CAG-EPO sequences, the pΔ28E4-CAG-EPO containsbacterial sequences of Kanamycin resistance and origin of replication,which are necessary for the DNA amplifying in bacteria (size 32.5 kb).

The construction of HDAd-EPO vector was as follows: In order to producethe HDAd-CAG-EPO (size 29.9 kb), the pΔ28E4-CAG-EPO plasmid was cut withthe PmeI restriction enzyme and the linearized vector was transfectedinto the 116 Cre-expressing producer cell line (see schematic of FIG.19). When the plasmid is linearized, the bacterial sequences wereremoved. The HDAd-EPO was then rescued by infecting the cells with thehelper virus AdNG163 containing a loxP-flanked packaging signal, whichresults in the excision of the viral packaging signal, rendering thehelper virus DNA un-packagable (Cre-mediated recombination between thetwo loxP sites of the HV results in ψ excision, rendering the helpervirus genome un-packagable). However, the helper virus will stillprovide all of the trans-acting factors necessary for replication andpackaging of pΔ28E4-CAG-EPO, which contains the appropriate cis-actingelements [i.e., viral packaging signal (ψ) and inverted terminal repeats(ITR)]. The titer of the HDAd was increased by serial co-infections(called passages) of 116 cells with the HDAd and the helper virus.

The non-replicating HDAd-EPO vector, is a key material used for thetissue processing, but was not directly administered to the subject. TheHDAd-EPO vector was produced in two stages:

1. Starting from the HDAd-EPO, whose derivation was detailed above,further vector batches are manufactured using the following steps:Amplification, in which the quantity of HDAd-EPO was increased by serialco-infections (passages) of the 116 producer cells with the HDAd-EPO andthe helper virus.

2. Large-scale production to generate large quantities of HDAd-EPO.

The HDAd vector was produced by co-infection of the 116 cells withAdNG163Helper Virus and HDAd-EPO vector in 10-layer Cell Factories.After a growth period with one media exchange, the 116 cells wereharvested by centrifugation, lysed by 3 freeze/thaw cycles, and DNA/RNAwas digested with use of benzonase. HDAd-EPO viral particles wereseparated from the Helper virus particles and other host cell componentsby use of a CsCl gradient followed by two CsCl isopyenic runs.

The HDAd-EPO vector produced was analyzed, the results of which arepresented in Table 2 below:

TABLE 2 Protocol Test Number Specifications Results Safety MycoplasmaDetection 30200 Direct Negative Negative 30200 Indirect NegativeInconclusive on harvest; Negative on final filled vialsBiologics-Sterility Test, Immersion, USP/ 30744 No Growth Negative (nogrowth) 21 CFR 610.12 30744A Biologics-Sterility Test 30736 NoInhibition PASS (no Inhibition) (Bacteriostasis/Fungistasis), Immersion,USP/21 CFR 610.12 Bacterial Endotoxin Test 37653 <5 EU/mL <0.500 EU/mLIn vitro Assay for Adventitious Viral 30521 No evidence of No evidenceof viral Contamination: MRC-5, VERO and Hs68 viral contamination Cells(Extended Duration) contamination In vivo Assay for Viral Contamination30027 No evidence of No evidence of viral (USFDA) Inculation ofEmbryonated Hen viral contamination Eggs, adult and Newborn Micecontamination Detection of Human Parovirus B19 DNA 30761 NegativeNegative by Quantitative Polymerase Chain Reaction (qPCR): GLP Detectionof Human Cytomegalovirus 30705 Negative Negative (CMV) DNA byQuantitative Polymerase Chain Reaction (qPCR): GLP Detection of HumanEpstein-Barr Virus 30713 Negative Negative (EBV) DNA QuantitativePolymerase Chain Reaction (qPCR): GLP Detection of Simian Virus 40(SV40) by 30715 Negative Negative DNA by Quantitative Polymerase ChainReaction (qPCR): (GLP) Detection of Human T-cell Lymphotropic 30985Negative Negative Virus 1 and 2 (HTLV-1 and -2) RNA by ReverseTranscriptase Quantitative Polymerase Chain Reaction (RT-qPCR): (GLP)Detection of Human Herpesvirus 6, 30863 Negative Negative Variant A(HHV-6 A) and Human Herpesvirus 6, Variant B (HHV-6 B) DNA byQuantitative Polymerase Chain Reaction (qPCR): (GLP) Detection of HumanHerpesvirus 7 DNA 30361 Negative Negative by Quantitative polymeraseChain Reaction (qPCR): (GLP) Detection of Human Herpesvirus 8 DNA 30913Negative Negative by Quantitative polymerase Chain Reaction (qPCR):(GLP) Detection of Hepatitis B Virus (HBV) 30703 Negative Negative DNAby Quantitative Polymerase Chain Reaction (qPCR): (GLP) Detection ofHIV-1 RNA by Reverse 30635 Negative Negative Transcriptase PolymeraseTranscriptase Quantitative polymerase Chain Reaction (RT-qPCR): GLPDetection of HIV-2 RNA by Reverse 30770 Negative NegativeTranscriptase-Polymerase Chain Reaction (RT-qPCR): LightCyclerAmplification and Fluorescence Probe Hybridiztion (GLP) Detection ofHuman Hepatitis C Virus 30730 Negative Negative (HCV) RNA by ReverseTranscriptase Quantitative polymerase Chain Reaction (RT-qPCR): (GLP)Detection of Human adeno-Associated 30415 Negative Negative Virus Type1, 2, 3, 4, 6, 7, 8, 10, 11 (AAV-Pan) DNA by Quantitative PolymeraseChain Reaction (qPCR): GLP Detection of Adventitious Bovine 30236Negative Negative Viruses: Extended Screening for Non- Bovine Cell LinesGrowth in Bovine Serum or Products Detection of Adventitious Porcine30674 Negative Negative Viruses by 9 CFR Regulations: GLP Detection ofHepatitis A Virus (HAV) 30665 Negative Negative RNA by RT-qPCR PurityDetection of RCA 30698 <1 VP in 3 × 0 VP in 3 × 10{circumflex over( )}10 vp 10{circumflex over ( )}10 vp % Helper virus in HD-Ad-CAG-EPO39283 <0.5%* 0.013% final vialed product by qPCR (R & D) Residual Hostcell DNA by TaqMan 30699 <10 ng/150 μl <50 pg per 150 μl Technology: GLPResidual for 3 Amplicons Using Taqman 30369 <10 ng/150 μl PASS: 1523.58pg DNA per Technology: GLP 150 μl for the 102 bp amplicon. PASS: 706.68pg DNA per 150 μl for the 401 bp amplicon. PASS: 623.75 pg DNA per 150μl for the 765 bp amplicon. Immunoenzymetric assay for the 38068 ReportPASS: <1 ng/ml of residual Determination of HEK 293 Host Cell HEK HCP,below the limit of Proteins the assay Immunological Detection ofBenzonase 38067 Report PASS: <1.25 ng/ml of Endonuclease Benzonase ®endonuclease, below the limit of the assay Fluorescent Polymerase ChainReaction 30357 Negative Negative (PCR) - based reverse transcription (F-PBRT) assay: (GLP) Strength Adenovirus Particle Number by HPLC 30783Report PASS: >1.4 × 10{circumflex over ( )}12 VP/ml Identity andPotency/Activity DNA Sequencing 30903 Report Confirmed as anticipatedGene Expression Protein >200 IU/bp/day In house tests show 700expression IU/BP/day on average following in vitro transduction OtherDetermination of pH and Appearance 38026 Clear, colorless Clear,colorless solution. No (GMP) visible particulates present.HDAd Vector Stability

In order to check vector stability, vector was aliquoted and stored at−80° C. for 17 months. At different time-points, one aliquot was thawedand 4 micro-organs (freshly harvested from human tummy tuck slabs) weretransduced with the same protocol and the same vector titer used toproduce genetically modified micro-organs. EPO production per day wasassessed during a period of 12-17 days post transduction for potencyassessment of the HDAd vector. In a retrospective analysis, the range ofEPO secretion per day (average of the 4 Biopumps on days 12-17) wasreviewed for each such experiment and they were grouped by the number ofmonths since the preparation of the frozen aliquots: 1-2 months, 6-9months, and 15-17 months post-aliquoting. The results are summarized inTable 3 below.

TABLE 3 HDAd-EPO lot#1 Test groups of 4 Biopumps were transduced with3.4 × 10{circumflex over ( )}10 VP per BP, and EPO production measureddays 12-17 Post-production time of Number of Avg IU/BP/day experiments:time from experiments Highest Lowest vector production in months. ingroup obtained obtained Months 1-2 7 721.6 214.5 Months 6-9 9 634.3246.2 Months 15-17 2 1201.7 355.4

Results from the table above suggested that there was no evidence ofreduced potency in genetically modified micro-organs prepared 15-17months after production of the vector.

Transduction Efficiency

The transduction efficiency with the HDAd-EPO vector is presented inFIG. 22. FIG. 22 illustrates that increasing the virus particle titer inthe transduction solution, causes an increase in EPO secretion from thegenetically modified micro-organs. Note that when titer was doubled from1.25×10⁹ to 2.50×10⁹, and then again to 5.00×10⁹, the EPO concentrationin the media also proportionally doubled. This increase probably relatesto an increase in the number of cells transduced or to an increase inthe copy number of VP per cell, indicating improved transductionefficiency. When VP titer was increased above 1.5×10¹⁰ per Biopump, nosignificant increase in EPO secretion was detected (data not shown),suggesting that the system under the current conditions is close tooptimal.

Ex Vivo Micro-Organ Maintenance

Every 3-4 days, used production media was collected, and the level ofthe secreted recombinant protein and glucose level were assessed alongwith the viability of the biopumps. Fresh Production media was added tothe 24-well plate.

Secreted Protein Measurements

Human EPO (hEPO) and IFNα concentration and secretion levels wereassayed using an enzyme-linked immunosorbent assay (ELISA) kit(Quantikine human erythropoietin; R&D Systems; Human interferon alphaELISA kit, PBL Biomedical Laboratories), according to the manufacturer'sinstructions.

In general, use of a genetically modified dermal micro-organ expressingand secreting EPO required that measurements 8-10 days aftertransduction of a group of 10 transduced dermal micro-organs, showedsecretion levels exceeding 200 U/BP/day. FIG. 20 represents the resultsof a test for EPO daily production from skins of different donors on day8 after transduction, all of which secrete in excess of the thresholdvalue of 200 U/BP/day.

Glucose Measurements

Glucose metabolism is used as a non-destructive assay to determine invitro genetically modified micro-organ viability. Tissue glucoseconsumption was evaluated using either Sigma-Aldrich Corporation'sGAGO20 Glucose (GO) Assay Kit, according to manufacturer's instructionsand/or a Glucose Meter (Accu-Check Sensor/Performa, Roche orequivalent).

Hematocrit Measurements

Hematocrit levels were assayed using centrifugation using the referencemethod recommended by The National Committee for Clinical LaboratoryStandards (NCCLS), as is known in the art. To determine the hematocrit,whole blood in a tube was centrifuged at 10-15,000×g for 5 minutes topellet the red cells (called packed erythrocytes), and the ratio of thecolumn of packed erythrocytes to the total length of the sample in thecapillary tube was measured with a graphic reading device within 10minutes of centrifugation.

Hemoglobin Measurements

Hb concentration levels were measured as part of over all laboratoryhematology assessments, which measured Hb, hematocrit, leukocytes,erythrocytes, MCV, MCH, reticulocyte, and platelet count. For instance,an automated Seimens ADVIA platform may be used to measure the CompleteBlood Counts. Hb was evaluated by two methods, a standard cyanmethemoglobin colorimetric method and flow cytometry.

Micro-Organ Implantation-SCID

In some experiments, genetically modified or control micro-organs wereimplanted subcutaneously in Severe Combined ImmunoDeficiency (SCID) miceafter assaying tissue glucose consumption to ascertain that micro-organswere viable. Male and female SCID mice weighing around 25 grams wereanaesthetized with 140 μl of diluted Ketaset (ketamine HCl) (400 μlKetaset and 600 μl saline) and control or EPO-expressing micro-organswere implanted subcutaneously ten days following micro-organtransduction.

Micro-Organ Implantation-Human

During the clinical trials, genetically modified or control micro-organswere implanted subcutaneously or intradermally under local anesthesia inthe lower abdomen, abdominal wall or the upper or lower back of the samehuman patients from which they were derived, after a patient screeningperiod which included signing the informed consent, laboratory tests,medical history, physical examination, ECG, and concomitant medicationdocumentation. In the results shown herein for the human clinicaltrials, day “0” is the day of implantation. In addition, GMMO andcontrol micro-organs were assayed for tissue glucose consumption toascertain that micro-organs were viable. Control or EPO-expressingmicro-organs were implanted subcutaneously or intradermally.

Example 1 EPO and IFNα Levels Produced In Vitro by GMMOs

Micro-organs were prepared as described above and transduced with ahelper-dependent adenoviral vector expressing an optimized IFNα genelinked to a CAG promoter, as described above. GMMOs were then maintainedin culture, and the levels of IFNα produced were evaluated by ELISA.Optimized IFNα-expressing micro-organs produced greater than 1000 ng/dayof IFNα in vitro (FIG. 1) for at least 40 days post-harvesting, andrecombinant hEPO-expressing micro-organs produced greater than 1000ng/day of hEPO in vitro (FIGS. 2A-B) for at least 142 dayspost-harvesting.

GMMOs comprising a gutless adenovirus vector encoding optimized hEPOmaintained higher percentages of peak expression for more than 200 dayscompared to micro-organs comprising an adenovirus-5 vector encoding hEPO(FIG. 3). Micro-organs comprising a gutless adenovirus vector encodingoptimized hEPO also maintained a higher percentage of peak expressionfor a longer period of time than micro-organs comprising a gutlessadenovirus vector encoding non-optimized hEPO (FIG. 4). Finally,micro-organs comprising a gutless adenovirus vector encoding hEPOdownstream of a CAG promoter showed higher levels of hEPO expression,which grew more pronounced as a function of post-transduction day,compared to micro-organs comprising a gutless adenovirus vector encodinghEPO downstream of a CMV promoter (FIG. 5).

Example 2 EPO Levels Produced by Human EPO-Expressing GMMOs MaintainedIn Vitro and in Serum of Implanted SCID Mice

EPO-expressing micro-organs were prepared as described above. After atotal of nine days in culture, the amount of EPO produced permicro-organ was measured, and this value was used to determine that eachmouse was implanted with micro-organs expressing equivalent levels ofEPO. On the tenth day, two micro-organs were implanted subcutaneouslyinto each SCID mouse and on the first measurement taken after ten days,levels of hEPO measured in the serum of the SCID mice were significantlyabove baseline levels. The levels remained high at least 216 dayspost-implantation and significantly raised hematocrit levels in SCIDmice for at least 157 days (FIG. 6A). Non-implanted EPO-expressingmicro-organs produced from the same donor at the same time as theimplanted EPO-expressing micro-organs but maintained in vitrocontinuously maintained high levels of EPO production (FIG. 6B).Micro-organs transduced with vectors comprising optimized hEPO geneproduced higher levels of EPO than those transduced with recombinanthEPO gene both in vivo (FIG. 6A) and in vitro (FIG. 6B). Control SCIDmice implanted with non-EPO-producing micro-organs showed no increase ofserum EPO levels and no significant changes in hematocrit levels aftermicro-organ implantation compared to pre-implantation (FIG. 6A).Micro-organs comprising EPO-expressing adenovirus-5, which was used as apositive control, was used at a titer of 1:10 compared to a titer of1:100 for micro-organs comprising EPO-expressing optimized ornon-optimized gutless adenovirus.

Example 3 Increased Hemoglobin (Hb) Levels Produced by Implanting HumanEPO-Expressing GMMO—Human Clinical Trials—

Clinical Studies Overview

The clinical studies below enlisted anemic, pre-dialysis chronic kidneydisease (CKD stage 3-4) patients The CKD stage is based on MDRD-GER[Modification of Diet in Renal Disease (MDRD) Study equation forestimating Glomerular Filtration Rate (GFR) from serum creatinine].Patients participating were not iron deficient using measurements oftransferrin saturation % (TSAT %) and ferritin (ng/ml) as criteria.Patients were either naïve with respect to EPO-dependency or ifEPO-dependent, they are withdrawn from erythropoiesis stimulating agents(ESA) for a period of at least 4 weeks. The term “patient” as usedherein may also be referred to herein as a “subject”.

For determination of Hb baseline, a baseline Hb average of values wasdetermined from the previous 30 days in naïve participants or baselinewas determined from the projected nadir 100 days post last ESA injectionand if available compared to historical Hb values. FIGS. 11A and 11Bshow Hb values prior to implantation and the projected nadir. Hb valuesafter implantation are also shown. Basal measurements: RBC count, Hb,Hct (%), absolute reticulocytes count, serum EPO level (mU/ml), ironstatus on day −30, day −9, and the day of implantation (day 0) werecollected.

Assessment of efficacy was performed by measuring EPO levels, Hb andhematocrit, and taking reticulocytes counts three times per week for thefirst 2 weeks, thereafter twice per week until the sixth week, and onceweekly thereafter. Criteria for assessment included Hb response andduration of effect.

FIG. 15 presents Demographic data for participants in the studiesdescribed below.

Safety results of the clinical trials support the advantages of use ofgenetically modified micro-organs in a clinical setting. Specifically,safety results showed: (a) no adverse events in any patients treated;(b) the procedure was brief and well tolerated; (c) no evidence ofanti-EPO antibody formation (blood samples were tested prior to, during,and after treatment and no increase in EPO antibodies was observed as aresult of treatment); and (d) no serum EPO levels to date exceeded 60mU/ml.

Clinical Trial—Single Administration

Phase I clinical trials were performed in Israel, in which pre-dialysisanemic patients with chronic kidney disease (CKD), stage III and stageIV, were implanted with autologous hEPO-GMMOs of the sustained type (along-lasting therapeutic formulation) of the present invention. A singleimplantation treatment with GMMO-hEPO provided between 3 to greater than16 months of effective EPO therapy. Approval for the Phase I/II GMMOhEPO trial was approved by Israel's Ministry of Health and was conductedat the Hadassah Medical Center and the Sourasky Tel Aviv Medical Center.The clinical trial was conducted according to regulatory and clinicalstandards of the FDA, in order to facilitate later US based clinicaltrials.

Patients were treated at 18-25 IU/kg/day (low dose) or at 35-45IU/kg/day (mid dose) or at 55-65 IU/kg/day (high dose). FIG. 7 shows thecorrelation of Net Peak serum EPO rise over baseline to the EPO doseadministered (IU/kg/day).

Results showed that Hb levels (Hb) were maintained in the 10-12 g/dlrange (therapeutic window) in the majority of patients using low doseadministration of 18-25 IU/kg/day, mid dose administration of 35-45IU/kg/day or high dose administration of 55-65 IU/kg/day. FIGS. 8A, 8Band 8C illustrates that the sustained Hb response within the therapeuticwindow, persisted for between 1-24 months. This sustained Hb responseresult is independent of any injections of EPO.

Male and female patients ranging in age from 21-82 were included in thestudy.

Patient 1 was an EPO naïve patient. Clinical statistics and details ofEPO trial for Patient 1 include: 82 year old male; Stage 4 CKD patient;GFR 21 ml/min, adequate iron stores (Transferrin saturation 33%,ferritin 340 ng/ml); and received low EPO dose (18-25 IU/kg/day)—2hEPO-GMMO administered.

Comments on treatment included: No adverse events; good Hb response; andsatisfied with treatment though tired of frequent blood sampling andfollow up visits for the study protocol—requested early terminationafter 4.5 months.

FIG. 9 shows sustained Hb response in patient 1, for a period of 131days.

Patient 2 was an EPO dependent patient. Clinical statistics and detailsof EPO trial for Patient 2 include: 71 year old male, Stage 3 CKDpatient; EPO dependent for 18 months, received 6000 IU every 10 days,last administered 40 days prior to EPO treatment; GFR 57 ml/min,adequate iron stores (Transferrin saturation 26%, ferritin 105 ng/ml);received low EPO dose (18-25 IU/kg/day)—3 hEPO-GMMOs administered

Comments on treatment included: No adverse events; satisfied withtreatment; good Hb response; and successfully completed over 12 monthsof follow-up. Patient is physically active and requests re-enrollmentfor higher dose after current treatment.

FIG. 10A shows sustained Hb response was maintained within thetherapeutic window in patient 2 for two years. FIGS. 10B and 10C showreticulocyte count and serum EPO levels, respectively, over a period ofgreater than 200 days for patient 2.

Patient 3 was an EPO dependent patient. Clinical statistics and detailsof EPO trial for Patient 3 include: 58 year old male, Stage 4 CKDpatient; EPO dependent for 16 months, received 5000 IU once per week,last administered 39 days prior to EPO treatment; GFR 26 ml/min,adequate iron stores (Transferrin saturation 23%, ferritin 243 ng/ml);and received low EPO dose (18-25 IU/kg/day)—4 hEPO-GMMOs administered

Comments on treatment included: No adverse events; good Hb response:elevation of ˜2 gm/dl over baseline Hb level. The target level notsustained at current dose. Patient's inclusion in study was terminatedto receive supplemental EPO injections.

FIG. 11A shows sustained Hb response was maintained just under thetarget therapeutic window in patient 3 for 92 days.

Patient 4 was an EPO naïve patient. Clinical statistics and details ofEPO trial for Patient 4 include: 57 year old female, Stage 3 CKDpatient; EPO naïve; GFR 32 ml/min, adequate iron stores; and receivedlow EPO dose (18-25 IU/kg/day)—5 hEPO-GMMOs administered.

Comments on treatment included: No adverse events and good Hb response.

FIG. 12 shows sustained Hb response within the therapeutic window inpatient 4, for a period of 224 days.

Patient 5 was an EPO dependent patient. Clinical statistics and detailsof EPO trial for Patient 5 include: 51 year old female, Stage 4 CKDpatient, polycystic kidney disease; EPO dependent, received 60 μg ofAranesp once per 3 weeks, last administered 113 days prior to EPOtreatment; GFR 18 ml/min, adequate iron stores (Transferrin saturation39%, ferritin 371 ng/ml); and received low EPO dose (18-25 IU/kg/day)—6hEPO-GMMOs administered.

Comments on treatment included: No adverse events; transient Hbresponse, insufficient; and discontinued from trial on day 35

Patient 6 was an EPO dependent patient. Clinical statistics and detailsof EPO trial for Patient 6 include: 40 year old female, Stage 4 CKDpatient, polycystic kidney disease—candidate for transplant; EPOdependent for 6 weeks, received 2000 IU twice per week, lastadministered 29 days prior to EPO treatment; GFR 25 ml/min, adequateiron stores (Transferrin saturation 33%); and received low EPO dose(18-25 IU/kg/day)—4 hEPO-GMMOs administered.

Comments on treatment included: No adverse events; good initial Hbresponse; deterioration in renal function; supplemental EPO injectionsgiven. Patient discontinued from study to go on dialysis.

FIG. 13 shows sustained Hb response within the therapeutic window inpatient 6, for a period of 89 days.

Patient 7 was an EPO naïve patient. Clinical statistics and details ofEPO trial for Patient 7 include: 77 year old male, Stage 4 CKD patient;EPO naïve; GFR 19 ml/min, adequate iron stores; and received medium EPOdose (35-45 IU/kg/day)—8 hEPO-GMMOs administered.

Comments on treatment included: No adverse events and was a lowresponder.

FIG. 14 shows sustained Hb response mostly within the therapeutic windowin patient 7, for a period of 172 days.

Results of this study showed blood of patients receiving low and mediumdoses of hEPO-GMMOs show sustained Hb levels within the therapeuticwindow for an extended period of time. Further, patients had no serious,product related adverse events and did not report major discomfortduring or after the procedures.

Clinical Trial—Dose Augmented by a Second Administration

Patient 10 was an EPO dependent patient treated with two low-doseadministrations. The first administration was at day 0 followed by asecond administration at day 70. FIG. 16 illustrates that the first dosesustained elevation of EPO at between 2-5 mU/ml above the baseline of 10mU/ml and Hb at 10.2 g/dl versus a nadir of 8.8 g/dl. Following theadministration of the second dose a similar peak response was observedin EPO serum levels and reticulocytes, with an apparent increase in Hb.

Results with a second administration show that retreatment is feasible,safe and effective.

Safety Results

This study was initiated in August 2008 and has been conducted at twoclinical sites. It was a Phase I/II, open label, single center,uncontrolled, dose escalation study, comprising three dosage groups(approximately 20, 40, and 60 EPO IU/kg/day). Table 4 below listsindividual demographics, dose, and previous EPO exposure of each of the17 patients treated through May 8, 2011.

TABLE 4 Target Age, EPO Weight dose [IU/ Actual dose Biopumps PatientSex naïve [kg] kg/day] [IU/kg/day] implanted  1 82, M Yes 84 18-25 21.42  2 72, M No 75 18-25 25.0 3  3 58, M No 84 18-25 24.7 4  4 68, F Yes62 18-25 23.5 5  5 50, F No 70 18-25 24.9 6  6 42, F No 75 18-25 23.4 4 7 77, M Yes 85 35-45 40.7 8  8 48, F Yes 62 35-45 45.1 3  9 76, M No 6735-45 45.0 4 10 (a) 64, F No 88 18-25 25.2 6 10 (b) 18-25 23.8 3 11 70,F No 98 35-45 43.7 5 12 21, F No 82 35-45 32.8 8 13 52, M No 70 35-4541.6 9 14 52, M Yes 53 55-65 64.7 3 15 60, F No 84 — — — 16 73, F Yes 8755-65 57.2 9 17 69, M No 58 55-65 62.0 9 18 44, F No 60 55-65 65.1 7

The patient procedures of harvesting and implantation have been welltolerated by the patients in the trial, for doses up to 65 U/kg/day.Erythema and edema have been noticed in some of the cases, andsubcutaneous hematoma was usually mild, with no observable correlationto the dose. Signs of the local subcutaneous hematoma disappear afterapproximately two weeks after the procedure. Local pain, reported insome cases, was usually mild and lasted only one day or slightly longerif no analgesic was taken.

Summary

Clinical trials presented show that hEPO-GMMO is safe and treatments areamenable to varying dosages, with feasibility demonstrated at dosages ofabout 18-25 IU/kg/day, 35-45 IU/kg/day and 55-65 IU/kg/day.Surprisingly, a single administration of an hEPO-GMMO provided asustained Hb response in the therapeutic window (10-12 g/dl blood Hb)for at least 3 months to more than 24 months for most patients.

Repeat administration had been shown to be safe and efficacious. At thesame time, it has been shown here that subcutaneous implantation of anhEPO-GMMO provided a sustained source of EPO for subjects in needthereof. Thus, subcutaneous implantation of hEPO-GMMO is a significantadvance in providing an effective sustained treatment alternative topatients in need, compared to months of frequent bolus injections ofESAs.

Example 4 Preclinical Toxicology and Physiological Function Studies inSCID Mice

A Good Laboratory Practice (GLP) toxicology study was performed atHarlan Biotech Israel. The objective of this study was to assess thepotential toxic effects of the Test Product Biopump HDAd-hEPO followinga single subcutaneous implantation in the NOD-SCID mouse for a maximalexposure period of 12 weeks in respect to its intended use as a constantsource for prolonged drug delivery in chronic renal failure disease.

The design of the toxicology study was reviewed in detail and agreedupon with the Head of FDA's Preclinical Division, and implementedaccordingly.

In view of the relatively short lifespan of SCID mice (ca 18 mo), thedurations presented in Table 5 were judged reasonable for testingtoxicological safety to support the proposed clinical trial.

TABLE 5 Experimental Design # Total # # sacrificed # sacrificedsacrificed BP Dose of mice @ 2 wks @ 8 wks @ 12 wks 300-450 IU 30 (15M,15F) 5M, 5F 5M, 5F 5M, 5F Epo/day Control - non 18 (9M, 9F) 3M, 3F 3M,3F 3M, 3F transduced Control - no 18 (9M, 9F) 3M, 3F 3M, 3F 3M, 3Fmicro-organ

In the first group, each animal received a portion of a Biopump (GMMO)that secreted in the range of 300-450 IU EPO/day.

The second group was an implanted control group, in which each animalreceived a similar sized portion of a dermal non-transduced micro-organ.

The third group as a “no micro-organ” control group, in which eachanimal was treated with identical subcutaneous related procedures as inthe control group but with no implant

Clinical Signs:

Throughout the entire observation period, careful clinical examinationswere carried out and recorded for all the animals in the study at leastonce daily. Observations include changes in skin, fur, eyes, mucousmembranes, occurrence of secretions and excretions (e.g. diarrhea) andautonomic activity. Changes in gait, posture and response to handling,as well as the presence of bizarre behavior, tremors, convulsions, sleepand coma were also observed and were recorded.

Body Weights:

Determination of individual body weights of all the animals in the studywas carried out at the randomization procedure, followed by body weightdetermination prior to the single subcutaneous implantation/shamoperation, 2 days later and thereafter on a weekly basis.

Food Consumption:

Measurements of food consumption was carried out during the acclimationperiod, followed by weekly basis measurements throughout the entireobservation period for all the animals in the study.

Collection of Blood Samples (Interim Bleeding Sessions):

All animals were subjected to interim bleeding sessions for Hematology &Biochemistry parameters, carried out every 13-15 days from the day ofthe single subcutaneous implantation. In order to keep the uniformitywithin the study, all the animals in the study were subjected to theinterim bleeding sessions. The blood samples served, among others, forthe purpose of measuring the Hematocrit (HCT) levels.

Necropsy Procedures & Macroscopic Examination:

All the animals originally assigned to the potential toxicityassessments were subjected to a full detailed necropsy and grosspathological examination.

Organ/Tissue Collection, Weighing & Fixations:

All the organs/tissues were collected during the respective schedulednecropsy session.

Results:

A test Product-treated female mouse was found dead in its cage on Day 7of the study. In addition, a Test Product-treated male mouse was founddead on Day 52. No further mortality occurred in any of the TestProduct, Control Item or Sham-operated groups (including spares).

No obvious treatment-related reactions were observed among the TestProduct, Control Item or Sham-operated surviving animal throughout theentire observation period.

Elevated HCT levels were recorded throughout the entire observationperiod within the Test Product—treated animals in view of the continuoussecretion of hEPO by the Test Product itself. Statistically significantincreased (p, 0.05 & p, 0.01) HCT values were revealed within the TestProduct—treated group on Days 15, 28, 43, 57 & 70 vs. the respectiveSham-Operated mean group values.

In view of the histopathological findings obtained under the conditionsof this study, it can be concluded that the Test Product BiopumpHDAd-hEPO, subcutaneously implanted in the NOD-SCID mouse as a constantsource for prolonged drug delivery at the dose of 300-450 IU/animal/dayfor a maximal exposure period of 12 weeks, is associated only withpharmacological-related changes, observed in all three scheduledtermination time points.

Further, long-term erythrostimulatory activities of Biopump HDAd-hEPO intwo comparable groups of SCID mice were run in parallel at two wellestablished animal facilities. Four mice of each group were implantedsubcutaneously with two Biopump HDAd-hEPO per mouse, secreting a totalof approximately 300 IU/day. The mice were bled every 10 days andhematocrit was measured by the centrifugation method and serum EPOlevels using a clinical grade EPO ELISA kit (note: EPO levels in themice serum were measured by an ELISA kit which is specific to human EPO,with no cross-reactivity to mice EPO). As seen in the FIG. 24, elevatedserum EPO levels and subsequent rise in hematocrit were found in bothexperimental groups, which maintained high levels of hematocrit forseveral months. The control mice did not have elevated serum EPO levelsor elevated hematocrit (data not shown).

Overall, these studies showed long-term elevation of serum EPO andelevation of hematocrit for up 8 months, which comprises a significantportion of the typical lifespan of the SCID mice.

Example 5 Long Term Ex Vivo EPO Secretion

Dermal micro-organs were prepared as described above and transduced witha helper-dependent adenoviral vector comprising SEQ ID No. 11 andexpressing EPO. The dermal GMMOs were then maintained in culture, andthe levels of EPO produced were evaluated by ELISA.

Results:

The bar graph presented in FIG. 23 shows long term ex-vivo EPO secretionafter transduction. Dermal GMMOs produced from dermis of two differentsubjects were monitored for over 6 months. In both cases, steadysecretion was observed, attesting to the continued presence of thetransgene, its structural stability and long-lasting consistentexpression of EPO.

Example 6 Preclinical Cell Count and Viability Cell Count Studies

The dermal micro-organ samples were maintained ex-vivo under cultureconditions which ensured optimal viability and wherein the dermalmicro-organ stays intact throughout the ex-vivo process.

To confirm that dermal micro-organs remain intact and viable and toassess the typical number of cells that can be expected in a geneticallymodified dermal micro-organ, sample genetically modified dermalmicro-organs were broken down into individual cells in order to countthe number of viable cells. The genetically modified dermal micro-organswere broken down by collagenase digestion followed by trypan bluestaining at day 1 and after 9 days of maintenance. The results showed nosignificant change in the number of cells during the maintenance period(FIG. 21 and Table 6), with approximately 1 million viable cells in eachgenetically modified dermal micro-organ.

TABLE 6 NUMBER OF CELLS (×10{circumflex over ( )}6)/BIOPUMP Days fromExperiment Experiment Experiment Experiment Experiment Experimenttransduction H-36 H-37 H-38 H-39 H-40 H-42 1 1.15 0.895 0.787 0.89 0.871.39 9 1.3 1.06 0.753 0.61 1.058 1.025

Since the micro-organs were transduced ex-vivo and the geneticallymodified micro-organs undergo several media exchanges during the ex-vivoprocessing over the course of 9-10 days, the residual viral load to thepatient was very low. Testing of the residual vector concentration inthe tissue before implantation showed 4 logs reduction below the initialvirus load in the transduction fluid (approximately 2.36 E10/BP fortransduction. In support, the clinical trial presented in Example 3demonstrated no patient safety issues were encountered in relationshipto the HDAd EPO vector.

Example 7 Phase II Human Clinical Trials

Clinical Study Overview

This is a Phase II, open-label, multi-center, controlled, randomized,tailored and titrated dose study. The trial is enrolling about 100candidate subjects (randomized into treatment or control groups) atapproximately 5-10 medical centers.

Subjects in the 2 arms undergo similar study evaluations. Each patientrandomized for treatment with dermal GMMO-hEPO undergoes harvesting of15 dermal tissue biopsies (micro-organs) from the lower abdomen underlocal anesthesia. The required portion of these micro-organs areprocessed and sent back to the treatment facility within 14 days ofharvesting for subcutaneous implantation back to the patient, also inthe lower abdomen under local anesthesia. The number of micro-organsprocessed into dermal GMMO-hEPO is based on the targeted initial doserequired by the patient derived from the dosage of previous EPOinjections. Unprocessed micro-organs are cryo-preserved for possiblelater use if additional EPO is required by the patient during thetitration period, defined as the first two months after initialadministration.

During the titration period, if the hemoglobin consistently declinesduring the first month after treatment, additional dermal GMMO-hEPO areprepared from cryo-preserved micro-organs in order to perform a secondadministration during the two-month titration period. The number ofdermal GMMO-hEPO to prepare is based on the rate of decline ofhemoglobin and clinical judgment.

In the case of excessive response, defined as hemoglobin rise above 12g/dl for 4 consecutive weeks, dose reduction by excision of one or moredermal GMMO-hEPO is performed. Dermal GMMO-hEPO are removed in order todecrease the total administered dose (in U) to the patient based on therate of elevation of the hemoglobin and clinical judgment. Furtherdermal GMMO-hEPO excision can be performed at any period if furtherexcessive response is observed in the patient.

The efficacy assessment period commences either after the doseadjustment (second administration of dermal GMMO-hEPO or resection ofdermal GMMO-hEPO), if performed, or from the initial administration. Theduration of the efficacy assessment period is 4 months. Following the 4months of efficacy assessment period, an additional period of 4 monthsof follow-up commences, during which the patient is further monitoredfor longer-term safety and duration of hemoglobin maintenance.

The objective of the trial is to maintain the patient's hemoglobinlevels in the range of 9-11 g/dl for 4 months or more with significantreduction in the need for erythropoietin-stimulating agent (ESA)injections. Laboratory parameters are used to evaluate the response ofthe treatment. Measurements blood parameters include complete bloodcount, EPO levels (by clinical ELISA kit), reticulocytes levels (aninitial sign of bone marrow response) and hemoglobin levels.

Patients who are in the control arm continue to receive their regularESA injections per the standard of care and will be evaluated similarlyto the treatment arm.

Clinical Trial

Subjects:

A total of about 100 human subjects are in this study. Two-thirds of thesubjects will be in the dermal GMMO-hEPO treatment group, and one-thirdof the subjects will be in the control arm that is treated by Standardof Care. Subjects may include end stage renal disease patients ondialysis.

Inclusion Criteria include at least: Adult male or female subjectsbetween 18 to 75 years of age at the time of screening visit; Subjectdiagnosed with anemia due to chronic renal failure CKD stage 5 and beingtreated with hemodialysis for at least 3 months; Patients treated withrecombinant erythropoietin to treat their anemia for at least 3 months;Stable Hb and EPO levels due to ESA injections, e.g., patient receivingESA injection therapy for at least 3 months, wherein hemoglobin levelshave been stable at physiological levels for at least one month;Efficient dialysis defined as: Kt/V>1.2; Subjects who are clinicallystable; Serum albumin >3.5 g/dl; 8. Subjects with adequate iron stores(transferrin saturation >20.0% and/or ferritin >200 ng/mL); and Willingto provide written informed consent to participate in the study.

Subjects requiring more than 65 IU/kg/day are excluded from the study.

Harvesting:

Fifteen dermal tissue samples (1.5-2.5 mm in diameter and approximately30 mm long) are harvested from the lower abdomen under local anesthesia.The harvest procedure utilizes a vacuum positioning device and asymmetrically sharpened coring needle attached to a commerciallyavailable medical drill. The tissue samples are primarily dermis(containing approximately 1 million fibroblasts on average) withpotentially trace amounts of epidermis and fat. The tissue samplesremain intact (are not broken down into individual cells) throughout theentire ex-vivo processing and even after implantation back into the samepatient.

The harvest process is performed only once. The initial administrationof dermal GMMO-hEPO is based on the previous dosage of EPO that thepatient received by injections prior to dermal GMMO-hEPO treatment.Micro-organs that are not processed into dermal GMMO-hEPO arecryo-preserved. If the initial dosage by dermal GMMO-hEPO is found to beinsufficient during the titration period (defined as the first twomonths after administration) further dermal GMMO-hEPO will be processedfrom the cryo-preserved micro-organs. A second administration will beperformed after the processing.

Implantation:

The micro-organ tissue samples (containing approximately 1 million cellson average) remain intact throughout the ex-vivo processing, includingthe transduction with the HDAd-EPO vector. These same tissue samples arethen administered back to the patient.

The implantation procedure utilizes a vacuum positioning device and animplantation needle. Each dermal GMMO-hEPO is loaded into the cannula ofthe implantation needle, and the needle is inserted subcutaneously,guided by the vacuum device, in the area of the lower abdomen underlocal anesthesia. A maximum of 15 dermal GMMO-hEPO can be implanted,depending on required dose of the patient being treated, which would bea total of approximately 15 million cells.

A second administration of dermal GMMO-hEPO may be performed during thetitration period, defined as the first two months after initialadministration, based on the hemoglobin response of the patient todermal GMMO-hEPO.

Analysis:

Each dermal GMMO-hEPO is characterized for daily secretion rate of EPO,and glucose consumption, ex vivo, which gives an indication of tissueviability. In order to assess the presence and effects of contaminants,during ex vivo maintenance, the spent medium is assayed for sterility,gram stain, mycoplasma and endotoxin. Individual dermal GMMO-hEPO willbe rejected if they fail the release criterion.

In addition, EPO concentration in the patient's serum is measured priorto and after implantation in order to determine elevation of serum EPOlevel above baseline after dermal GMMO-hEPO administration. Follow-upand measurements will be for between seven and thirteen months from thelast of implantation date.

Results:

It is expected that patients will show maintenance of hemoglobin withinthe range of 9-11 g/dl for 4 months or more with a single administrationof dermal GMMO-hEPO, adjusted to dose, with significant reduction in theneed for ESA injections.

Secondarily, it is expected that implantation will result in theavoidance of supra-physiological elevations of serum EPO levels definedas above 200 mU/ml, and reduction of exogenous EPO administration incomparison to standard of care (SOC).

Measurement of the hemoglobin level is the primary indication of theprogression of the anemia. The hemoglobin levels of the patients aremonitored on a weekly basis throughout the treatment. In addition,laboratory parameters are used to evaluate the response of thetreatment. Measurements are of blood parameters including complete bloodcount, EPO levels, and reticulocytes levels (an initial sign of bonemarrow response).

Patients in the trial will be in the study for about 10 months afterinitial administration of dermal GMMO-hEPO.

Summary:

The major anticipated clinical benefit of the clinical trial is themaintenance of the hemoglobin within the target therapeutic window of9-11 g/dl with significantly reduced need for additional injections ofESAs and/or blood transfusions. It is anticipated that a continuoussteady delivery of EPO will enable better hemoglobin control incomparison to EPO injections and will result in less hemoglobin cyclingwhich has been linked to increased morbidity and mortality in renalanemia patients.

It will be appreciated by a person skilled in the art that the presentinvention is not limited by what has been particularly shown anddescribed hereinabove. Rather, the scope of the invention is defined bythe claims that follow:

What is claimed is:
 1. A method of treating anemia in a human subject inneed over a sustained time period comprising the steps of: (a) providingat least one autologous erythropoietin secreting genetically modifieddermal micro-organ that comprises a helper-dependent adenovirus vectorcomprising the nucleic acid sequence encoding erythropoietin operablylinked to one or more regulatory sequences, wherein said nucleic acidcomprises SEQ ID NO: 11, (b) determining the erythropoietin secretionlevels of the at least one genetically modified dermal micro-organ instep (a) in vitro, (c) implanting the autologous genetically modifieddermal micro-organ of step (b) in said human subject at an effectivedosage at a site selected from the group consisting of subcutaneously,subdermally and intradermally, (d) measuring hemoglobin levels in theblood of said human subject from step (c); and (e) maintaininghemoglobin levels in said human subject at 9-11 g/dl in at least 50% ofthe measurements for at least one month, thereby treating anemia in ahuman subject.
 2. The method of claim 1, wherein the autologousgenetically modified dermal micro-organ to be implanted provides said aneffective dosage of 18-150 U erythropoietin/Kg body weight of saidsubject/day, as determined by in vitro secretion of erythropoietin fromthe genetically modified micro-organ.
 3. The method of claim 2, whereinthe autologous genetically modified dermal micro-organ to be implantedprovides said an effective dosage of 18-30 U erythropoietin/Kg bodyweight of said subject/day, as determined by in vitro secretion oferythropoietin from the genetically modified micro-organ.
 4. The methodof claim 2, wherein the autologous genetically modified dermalmicro-organ to be implanted provides said an effective dosage of 30-50 Uerythropoietin/Kg body weight of said subject/day, as determined by invitro secretion of erythropoietin from the genetically modifiedmicro-organ.
 5. The method of claim 2, wherein the autologousgenetically modified dermal micro-organ to be implanted provides said aneffective dosage of 50-65 U erythropoietin/Kg body weight of saidsubject/day, as determined by in vitro secretion of erythropoietin fromthe genetically modified micro-organ.
 6. The method of claim 2, whereinsaid effective dosage is determined based on: a. said subject′s weight;b. said subject′s historical hemoglobin levels; and c. the averageamount of erythropoientin administered to said subject in the one monthprior said implanting step.
 7. The method of claim 1, wherein saidmeasured hemoglobin levels are 9-11 g/dl in at least 50% of themeasurements for at least six months.
 8. The method of claim 1, furthercomprising a step of implanting at a later date to said human subject,at least one additional autologous erythropoietin secreting geneticallymodified dermal micro-organ that comprises a helper-dependent adenovirusvector comprising the nucleic acid sequence encoding erythropoietinoperably linked to one or more regulatory sequences, wherein saidnucleic acid comprises SEQ ID NO:
 11. 9. The method of claim 1, furthercomprising a step of maintaining said at least one genetically modifieddermal micro-organ in vitro for at least 9 days, prior to saidimplantation step.
 10. The method of claim 1, wherein said subject isselected from a group consisting of: a. a subject suffering from: renalfailure, chronic renal failure, chemotherapy induced anemia, anemia as aresult of HIV treatments, microangiopathic haemolytic anemia, anemia asa result of prematurity, an inflammatory condition including rheumatoidarthritis, an infection, anemia associated with cancers includingmultiple myeloma and non-Hodgkin lymphoma, hematopoietic stem celldisorders, sickle cell anemia or thalassemia; and b. a subject in needof accelerated erythroid repopulation after bone marrow transplantation;or any combination thereof.
 11. The method of claim 10, wherein saidsubject suffering from chronic renal failure is suffering from chronickidney disease (CKD) or end stage renal disease (ESRD).
 12. A method ofincreasing or maintaining physiological hemoglobin levels in a humansubject in need over a sustained time period comprising the steps of:(a) providing at least one autologous erythropoietin secretinggenetically modified dermal micro-organ that comprises ahelper-dependent adenovirus vector comprising the nucleic acid sequenceencoding erythropoietin operably linked to one or more regulatorysequences, wherein said nucleic acid comprises SEQ ID NO: 11, (b)determining the erythropoietin secretion levels of the at least onegenetically modified dermal micro-organ in step (a) in vitro, (c)implanting the autologous genetically modified dermal micro-organ ofstep (b) in said human subject at an effective dosage at a site selectedfrom the group consisting of subcutaneously, subdermally andintradermally, (d) measuring hemoglobin levels in the blood of saidhuman subject from step (c); and (e) maintaining hemoglobin levels insaid human subject at 9-11 g/dl in at least 50% of the measurements forat least one month, thereby increasing or maintaining physiologicalhemoglobin levels in a human subject.
 13. The method of claim 12,wherein the autologous genetically modified dermal micro-organ to beimplanted provides said an effective dosage of 18-150 Uerythropoietin/Kg body weight of said subject/day, as determined by invitro secretion of erythropoietin from the genetically modifiedmicro-organ.
 14. The method of claim 13, wherein the autologousgenetically modified dermal micro-organ to be implanted provides said aneffective dosage of 18-30 U erythropoietin/Kg body weight of saidsubject/day, as determined by in vitro secretion of erythropoietin fromthe genetically modified micro-organ.
 15. The method of claim 13,wherein the autologous genetically modified dermal micro-organ to beimplanted provides said an effective dosage of 30-50 U erythropoietin/Kgbody weight of said subject/day, as determined by in vitro secretion oferythropoietin from the genetically modified micro-organ.
 16. The methodof claim 13, wherein the autologous genetically modified dermalmicro-organ to be implanted provides said an effective dosage of 50-65 Uerythropoietin/Kg body weight of said subject/day, as determined by invitro secretion of erythropoietin from the genetically modifiedmicro-organ.